Leafhopper ecdysone receptor nucleic acids, polypeptides, and uses thereof

ABSTRACT

The present invention relates to a novel isolated leafhopper ecdysone receptor polypeptide. The invention also relates to an isolated nucleic acid encoding the leafhopper ecdysone receptor polypeptide, to vectors comprising them and to their uses, in particular in methods for modulating gene expression in an ecdysone receptor-based gene expression modulation system and methods for identifying molecules that modulate leafhopper ecdysone receptor activity.

FIELD OF THE INVENTION

This invention relates to the field of biotechnology. Specifically, thisinvention relates to isolated nucleic acids, vectors comprising them,and polypeptides encoded by them, and to their use in the field of geneexpression and insecticide discovery. More specifically, this inventionrelates to a novel nucleic acid encoding an ecdysone receptorpolypeptide from the homopteran green leafhopper (Nephotetix cincticeps,∫NcEcR”) and its use in methods of modulating the expression of a genewithin a host cell using NcEcR, and in methods of identifying moleculesthat modulate the activity of the NcEcR.

BACKGROUND OF THE INVENTION

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties. However, the citation ofany reference herein should not be construed as an admission that suchreference is available as “Prior Art” to the instant application.

Cultivated agriculture has greatly increased efficiency of foodproduction in the world. However, various insect pests have found itadvantageous to seek out and exploit cultivated sources of food to theirown advantage. These insect pests typically develop by a temporalsequence of events which are characteristic of their order. Many insectsinitially develop in a caterpillar or maggot-like larval form.Thereafter, they undergo a significant metamorphosis from which an adultemerges having characteristic anatomical features. Anatomic similarityis a reflection of developmental, physiological and biochemicalsimilarities shared by these creatures. In particular, the principles ofthe insect ecdysteroid-hormone receptors and development, as describedby Ashburner et al. (Cold Spring Harbor Symp. Quant. Biol. 38: 655-662,1974), likely would be shared by many different types of insects.

To prevent or reduce the destruction of cultivated crops by insects,organic molecules with pesticidal properties are used commonly inattempts to eliminate or reduce the insect populations.

However, the ecological side effects of these pesticides, due in part totheir broad activity and lack of specificity, and in part, to the factthat some of these pesticides are not easily biodegradable,significantly affect populations of both insect and other species ofanimals. Some of these organisms may be advantageous from an ecologicalor other perspective. Furthermore, as the insect populations evolve indirections to minimize the effects of the applied pesticides, theamounts of pesticides applied are often elevated so high as to causesignificant effects on other animals, including humans, which areaffected directly or indirectly by the application of the pesticides.Thus, an important need exists for both highly specific pesticides orhighly active pesticides which have biological effects only on thespecies of animals targeted by the pesticides, and are biodegradable.Novel insect hormones which, like the ecdysteroids, act by complexingwith insect members of the steroid receptor superfamily to controlinsect development, are likely candidates for pesticides with thesedesirable properties.

Growth, molting, and development in insects are regulated by theecdysone steroid hormone (molting hormone) and the juvenile hormones(Dhadialla, et al., 1998, Annu. Rev. Entomol. 43: 545-569). Themolecular target for ecdysone in insects consists of at least ecdysonereceptor (EcR) and ultraspiracle protein (USP). EcR is a member of thenuclear steroid receptor super family that is characterized by signatureDNA and ligand binding domains, and an activation domain (Koelle et al.1991, Cell, 67: 59-77). EcR receptors are responsive to a number ofsteroidal compounds such as ponasterone A and muristerone A. Recently,non-steroidal compounds with ecdysteroid agonist activity have beendescribed, including the commercially available insecticidestebufenozide and methoxyfenozide (see International Patent ApplicationNo. PCT/EP96/00686 and U.S. Pat. No. 5,530,028). Both analogs haveexceptional safety profiles to other organisms.

Polynucleotides encoding ecdysone receptors have been cloned from avariety of insect species, including Dipterans (see U.S. Pat. Nos.5,514,578 and 6,245,531 B1), Lepidopterans, Orthopterans, Hemipterans,and one Homopteran Aphid, all from the class Arthropod. In particular,EcRs have been cloned from spruce budworm Chonstoneura fumiferana EcR(“CfEcR”; Kothapalli et al., 1995 Dev Genet. 17: 319-30), a yellow mealworm Tenebrio molitor EcR (“TmEcR”; Mouillet et al., 1997, Eur. J.biochem. 248: 856-863), a tobacco hormworm Manduca sexta EcR (“MsEcR”;Fujiwara et al., 1995, Insect Biochem. Molec. Biol. 25, 845-856), atobacco budworm Heliothies virescens EcR (“HvEcR”; Martinez et al.,1999, Insect Biochem Mol Biol. 29: 915-30), a golmidge Chironomustentans EcR (“CtEcR”; Imhof et al., 1993, Insect Biochem. Molec. Biol.23: 115-124), a silkworm Bombyx mori EcR (“BmEcR”; Swevers et al., 1995,Insect Biochem Molec. Biol. 25: 857-866), a squinting bush brownBicyclus anynana EcR (“BanEcR”), a buckeye Junonia coenia EcR (“JcEcR”),a fruit fly Drosophila melanogaster EcR (“DmEcR”; Koelle et al., 1991,Cell 67: 59-77), a yellow fever mosquito Aedes aegypti EcR (“AaEcR”; Choet al., 1995, Insect Biochem. Molec. Biol. 25: 19-27), a blowfly Luciliacapitata (“LcEcR”), a sheep blowfly Lucilia cuprina EcR (“LucEcR”;Hannan and Hill, 1997, Insect Biochem. Molec. Biol. 27: 479-488), ablowfly Calliphora vicinia EcR (“CvEcR”), a Mediterranean fruit flyCeratitis capitata EcR (“CcEcR”; Verras et al., 1999, Eur J Biochem.265: 798-808), a locust Locusta migratoria EcR (“LmEcR”; Saleh et al.,1998, Mol Cell Endocrinol. 143: 91-9), an aphid Myzus persicae EcR(“MpEcR”; International Patent Application Publication WO99/36520), afiddler crab Celuca pugilator EcR (“CpEcR”; Chung et al., 1998, Mol CellEndocrinol 139: 209-27), and an ixodid tick Amblyomma americanum EcR(“AmaEcR”; Guo et al., 1997, Insect Biochem. Molec. Biol. 27: 945-962).The nucleotide and/or amino acid sequences of these ecdysone receptorshave been determined and are publicly available.

The ecdysone receptor complex typically includes proteins that aremembers of the nuclear receptor superfamily wherein all members aregenerally characterized by the presence of an amino-terminaltransactivation domain, a DNA binding domain (“DBD”), and a ligandbinding domain (“LBD”) separated from the DBD by a hinge region. As usedherein, the term “DNA binding domain” comprises a minimal polypeptidesequence of a DNA binding protein, up to the entire length of a DNAbinding protein, so long as the DNA binding domain functions toassociate with a particular response element. Members of the nuclearreceptor superfamily are also characterized by the presence of four orfive domains: A/B, C, D, E, and in some members F (see U.S. Pat. No.4,981,784 and Evans, Science 240:889-895 (1988)). The “A/B” domaincorresponds to the transactivation domain, “C” corresponds to the DNAbinding domain, “D” corresponds to the hinge region, and “E” correspondsto the ligand binding domain. Some members of the family may also haveanother transactivation domain on the carboxy-terminal side of the LBDcorresponding to “F”.

The DBD is characterized by the presence of two cysteine zinc fingersbetween which are two amino acid motifs, the P-box and the D-box, whichconfer specificity for ecdysone response elements. These domains may beeither native, modified, or chimeras of different domains ofheterologous receptor proteins. The EcR receptor, like a subset of thesteroid receptor family, also possesses less well-defined regionsresponsible for heterodimerization properties. Because the domains ofnuclear receptors are modular in nature, the LBD, DBD, andtransactivation domains may be interchanged.

Gene switch systems are known that incorporate components from theecdysone receptor complex. However, in these known systems, whenever EcRis used it is associated with native or modified DNA binding domains andtransactivation domains on the same molecule. USP or RXR are typicallyused as silent partners. Applicants have previously shown that when DNAbinding domains and transactivation domains are on the same molecule thebackground activity in the absence of ligand is high and that suchactivity is dramatically reduced when DNA binding domains andtransactivation domains are on different molecules, that is, on each oftwo partners of a heterodimeric or homodimeric complex (seePCT/US01/09050).

The insect ecdysone receptor (EcR) heterodimerizes with Ultraspiracle(USP), the insect homologue of the mammalian RXR, and binds ecdysteroidsand ecdysone receptor response elements and activates transcription ofecdysone responsive genes (Riddiford et al. 2000, Vitam Horm, 60:1-73).The EcR/USP/ligand complexes play important roles during insectdevelopment and reproduction. The EcR is a member of the steroid hormonereceptor superfamily and has five modular domains, A/B(transactivation), C (DNA binding, heterodimerization), D (Hinge,heterodimerization), E (ligand binding, heterodimerization andtransactivation and in some cases, F (transactivation), domains. Some ofthese domains such as A/B, C and E retain their function when they arefused to other proteins.

Recently, ecdysone receptor based gene expression systems have beendeveloped. Tightly regulated inducible gene expression systems or “geneswitches” are useful for various applications such as gene therapy,large scale production of proteins in cells, cell based high throughputscreening assays, functional genomics and regulation of traits intransgenic plants and animals. U.S. Pat. No. 6,265,173 B1 discloses thatvarious members of the steroid/thyroid superfamily of receptors cancombine with Drosophila melanogaster ultraspiracle receptor (USP) orfragments thereof comprising at least the dimerization domain of USP foruse in a gene expression system. U.S. Pat. No. 5,880,333 discloses aDrosophila melanogaster EcR and ultraspiracle (USP) heterodimer systemused in plants in which the transactivation domain and the DNA bindingdomain are positioned on two different hybrid proteins.

The first version of an EcR-based gene switch used Drosophilamelanogaster EcR (DmEcR) and Mus musculus RXR (MmRXR) and showed thatthese receptors in the presence of steroid, ponasteroneA, transactivatereporter genes in mammalian cell lines and transgenic mice(Christopherson et al. 1992, Proc. Natl. Acad. Sci. U.S.A. 89:6314-6318; No et al. 1996, Proc. Natl. Acad. Sci. 93: 3346-3351). Later,Suhr et al. (1998, Proc. Natl. Acad. Sci. U.S.A. 95: 7999-8004) showedthat non-steroidal ecdysone agonist, tebufenozide, induced high level oftransactivation of reporter genes in mammalian cells through Bombyx moriEcR (BmEcR) in the absence of exogenous heterodimer partner.

International Patent Applications Nos. PCT/US97/05330 (WO 97/38117) andPCT/US99/08381 (WO99/58155) disclose methods for modulating theexpression of an exogenous gene in which a DNA construct comprising theexogenous gene and an ecdysone response element is activated by a secondDNA construct comprising an ecdysone receptor that, in the presence of aligand therefor, and optionally in the presence of a receptor capable ofacting as a silent partner, binds to the ecdysone response element toinduce gene expression. The ecdysone receptor of choice was isolatedfrom Drosophila melanogaster. Typically, such systems require thepresence of the silent partner, preferably retinoid X receptor (RXR), inorder to provide optimum activation In mammalian cells, insect ecdysonereceptor (EcR) heterodimerizes with retinoid X receptor (RXR) andregulates expression of target genes in a ligand dependent manner.International Patent Application No. PCT/US98/14215 (WO 99/02683)discloses that the ecdysone receptor isolated from the silk moth Bombyxmori is functional in mammalian systems without the need for anexogenous dimer partner.

Unfortunately, these USP-based systems are constitutive in animal cellsand therefore, are not effective for regulating reporter geneexpression. Drawbacks of the above described EcR-based gene regulationsystems include a considerable background activity in the absence ofligands and non-applicability of these systems for use in both plantsand animals (see U.S. Pat. No. 5,880,333).

Recently, an improved ecdysone receptor-based inducible gene expressionsystem has been developed in which the transactivation and DNA bindingdomains are separated from each other by placing them on two differentproteins results in greatly reduced background activity in the absenceof a ligand and significantly increased activity over background in thepresence of a ligand (pending application PCT/US01/09050, incorporatedherein in its entirety by reference). This two-hybrid system is asignificantly improved inducible gene expression modulation systemcompared to the two systems disclosed in applications PCT/US97/05330 andPCT/US98/14215. The two-hybrid system exploits the ability of a pair ofinteracting proteins to bring the transcription activation domain into amore favorable position relative to the DNA binding domain such thatwhen the DNA binding domain binds to the DNA binding site on the gene,the transactivation domain more effectively activates the promoter (see,for example, U.S. Pat. No. 5,283,173). Briefly, the two-hybrid geneexpression system comprises two gene expression cassettes; the firstencoding a DNA binding domain fused to a nuclear receptor polypeptide,and the second encoding a transactivation domain fused to a differentnuclear receptor polypeptide. In the presence of ligand, the interactionof the first polypeptide with the second polypeptide effectively tethersthe DNA binding domain to the transactivation domain. Since the DNAbinding and transactivation domains reside on two different molecules,the background activity in the absence of ligand is greatly reduced.

A two-hybrid system also provides improved sensitivity to non-steroidalligands for example, diacylhydrazines, when compared to steroidalligands for example, ponasterone A (“PonA”) or muristerone A (“MurA”).That is, when compared to steroids, the non-steroidal ligands providehigher activity at a lower concentration. In addition, sincetransactivation based on EcR gene switches is often cell-line dependent,it is easier to tailor switching systems to obtain maximumtransactivation capability for each application. Furthermore, thetwo-hybrid system avoids some side effects due to overexpression of RXRthat often occur when unmodified RXR is used as a switching partner. Ina preferred two-hybrid system, native DNA binding and transactivationdomains of EcR or RXR are eliminated and as a result, these hybridmolecules have less chance of interacting with other steroid hormonereceptors present in the cell resulting in reduced side effects.

Applicants have now obtained and determined the full length codingsequence of an additional homopteran EcR polynucleotide for use inmethods of modulating gene expression in a host cell and methods ofidentifying molecules that modulate activity of leafhopper EcR. Asdescribed herein, Applicants' invention provides novel leafhopperecdysone receptor polypeptides and novel polynucleotides encoding thesepolypeptides that are useful as components of gene expression systemsfor highly specific regulation of recombinant proteins in host cells orin methods for identifying new molecules which may act as agonists orantagonists of a homopteran insect ecdysone receptor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Transactivation of reporter genes through VP16/NcEcR-CDEconstruct transfected into L57 cells or CBW cells along with 5XEcRELacZand pFREcRE by 20E or RH-123709. The numbers on top of the bars indicatefold increase over DMSO levels.

FIG. 2: Transactivation of reporter genes through GAL4/NcEcR-DEconstruct transfected into NIH3T3 cells along with VP16/CfUSP-EF,VP16/DmUSP-EF, VP16/MmRXRα-EF, VP16/MmRXRα/LmUSP-EF chimera,VP16/AmaRXR1-EF, or VP16/Ama2-EF, and pFRLuc by PonA or GS™-E. Thenumbers on top of the bars indicate the maxi-mum fold induction for thatgroup.

DETAILED DESCRIPTION OF THE INVENTION

The present invention advantageously provides an isolated polynucleotideencoding a novel leafhopper ecdysone receptor polypeptide. Thepolynucleotides and polypeptides of the present invention are useful inmethods to regulate gene expression of a polypeptide of interest in ahost cell and in identifying new molecules that modulate activity of aleafhopper EcR.

The various aspects of the invention will be set forth in greater detailin the following sections, directed to the nucleic acids, polypeptides,vectors, antibodies, compositions, and methods of use of the invention.This organization into various sections is intended to facilitateunderstanding of the invention, and is in no way intended to be limitingthereof.

DEFINITIONS

The following defined terms are used throughout the presentspecification, and should be helpful in understanding the scope andpractice of the present invention.

In a specific embodiment, the term “about” or “approximately” meanswithin 20%, preferably within 10%, more preferably within 5%, and evenmore preferably within 1% of a given value or range.

The term “substantially free” means that a composition comprising “A”(where “A” is a single protein, DNA molecule, vector, recombinant hostcell, etc.) is substantially free of “B” (where “B” comprises one ormore contaminating proteins, DNA molecules, vectors, etc.) when at leastabout 75% by weight of the proteins, DNA, vectors (depending on thecategory of species to which A and B belong) in the composition is “A”.Preferably, “A” comprises at least about 90% by weight of the A+Bspecies in the composition, most preferably at least about 99% byweight. It is also preferred that a composition, which is substantiallyfree of contamination, contain only a single molecular weight specieshaving the activity or characteristic of the species of interest.

The term “isolated” for the purposes of the present invention designatesa biological material (nucleic acid or protein) that has been removedfrom its original environment (the environment in which it is naturallypresent). For example, a polynucleotide present in the natural state ina plant or an animal is not isolated, however the same polynucleotideseparated from the adjacent nucleic acids in which it is naturallypresent, is considered “isolated”. The term “purified” does not requirethe material to be present in a form exhibiting absolute purity,exclusive of the presence of other compounds. It is rather a relativedefinition.

A polynucleotide is in the “purified” state after purification of thestarting material or of the natural material by at least one order ofmagnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.

As used herein, the term “substantially pure” describes a polypeptide orother material which has been separated from its native contaminants.Typically, a monomeric polypeptide is substantially pure when at leastabout 60 to 75% of a sample exhibits a single polypeptide backbone.Minor variants or chemical modifications typically share the samepolypeptide sequence. Usually a substantially pure polypeptide willcomprise over about 85 to 90% of a polypeptide sample, and preferablywill be over about 99% pure. Normally, purity is measured on apolyacrylamide gel, with homogeneity determined by staining.Alternatively, for certain purposes high resolution will be necessaryand HPLC or a similar means for purification will be used. For mostpurposes, a simple chromatography column or polyacrylamide gel will beused to determine purity.

The term “substantially free of naturally-associated host cellcomponents” describes a polypeptide or other material which is separatedfrom the native contaminants which accompany it in its natural host cellstate. Thus, a polypeptide which is chemically synthesized orsynthesized in a cellular system different from the host cell from whichit naturally originates will be free from its naturally-associated hostcell components.

The terms “nucleic acid” or “polynucleotide” are used interchangeablyherein to refer to a polymeric compound comprised of covalently linkedsubunits called nucleotides. Nucleic acid includes polyribonucleic acid(RNA) and polydeoxyribonucleic acid (DNA), both of which may besingle-stranded or double-stranded. DNA includes but is not limited tocDNA, genomic DNA, plasmids DNA, synthetic DNA, and semi-synthetic DNA.DNA may be linear, circular, or supercoiled.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenon-transcribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

The term “fragment” will be understood to mean a nucleotide sequence ofreduced length relative to the reference nucleic acid and comprising,over the common portion, a nucleotide sequence identical to thereference nucleic acid. Such a nucleic acid fragment according to theinvention may be, where appropriate, included in a larger polynucleotideof which it is a constituent. Such fragments comprise, or alternativelyconsist of, oligonucleotides ranging in length from at least 6-1100consecutive nucleotides of a nucleic acid according to the invention.

As used herein, an “isolated nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

A “gene” refers to an assembly of nucleotides that encode a polypeptide,and includes cDNA and genomic DNA nucleic acids. “Gene” also refers to anucleic acid fragment that expresses a specific protein or polypeptide,including regulatory sequences preceding (5′ non-coding sequences) andfollowing (3′ non-coding sequences) the coding sequence. “Native gene”refers to a gene as found in nature with its own regulatory sequences.“Chimeric gene” refers to any gene that is not a native gene, comprisingregulatory and/or coding sequences that are not found together innature. Accordingly, a chimeric gene may comprise regulatory sequencesand coding sequences that are derived from different sources, orregulatory sequences and coding sequences derived from the same source,but arranged in a manner different than that found in nature. A chimericgene may comprise coding sequences derived from different sources and/orregulatory sequences derived from different sources. “Endogenous gene”refers to a native gene in its natural location in the genome of anorganism. A “foreign” gene or “heterologous” gene refers to a gene notnormally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Heterologous” DNA refers to DNA not naturally located in the cell, orin a chromosomal site of the cell. Preferably, the heterologous DNAincludes a gene foreign to the cell.

The term “genome” includes chromosomal as well as mitochondrial,chloroplast and viral DNA or RNA.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., 1989 infra). Hybridization andwashing conditions are well known and exemplified in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor(1989), particularly Chapter 11 and Table 11.1 therein (entirelyincorporated herein by reference). The conditions of temperature andionic strength determine the “stringency” of the hybridization.

Stringency conditions can be adjusted to screen for moderately similarfragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. For preliminaryscreening for homologous nucleic acids, low stringency hybridizationconditions, corresponding to a T_(m) of 55°, can be used, e.g., 5×SSC,0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5%SDS). Moderate stringency hybridization conditions correspond to ahigher T_(m), e.g., 40% formamide, with 5× or 6×SCC. High stringencyhybridization conditions correspond to the highest T_(m), e.g., 50%formamide, 5× or 6×SCC.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The term “complementary” is usedto describe the relationship between nucleotide bases that are capableof hybridizing to one another. For example, with respect to DNA,adenosine is complementary to thymine and cytosine is complementary toguanine. Accordingly, the instant invention also includes isolatednucleic acid fragments that are complementary to the complete sequencesas disclosed or used herein as well as those substantially similarnucleic acid sequences.

In a specific embodiment of the invention, polynucleotides are detectedby employing hybridization conditions comprising a hybridization step atT_(m) of 55° C., and utilizing conditions as set forth above. In apreferred embodiment, the T_(m) is 60° C.; in a more preferredembodiment, the T_(m) is 63° C.; in an even more preferred embodiment,the T_(m) is 65° C.

Post-hybridization washes also determine stringency conditions. One setof preferred conditions uses a series of washes starting with 6×SSC,0.5% SDS at room temperature for 15 minutes (min), then repeated with2×SSC, 0.5% SDS at 45° C. for 30 minutes, and then repeated twice with0.2×SSC, 0.5% SDS at 50° C. for 30 minutes. A more preferred set ofstringent conditions uses higher temperatures in which the washes areidentical to those above except for the temperature of the final two 30min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Anotherpreferred set of highly stringent conditions uses two final washes in0.1×SSC, 0.1% SDS at 65° C.

The appropriate stringency for hybridizing nucleic acids depends on thelength of the nucleic acids and the degree of complementation, variableswell known in the art. The greater the degree of similarity or homologybetween two nucleotide sequences, the greater the value of T_(m) forhybrids of nucleic acids having those sequences. The relative stability(corresponding to higher T_(m)) of nucleic acid hybridizations decreasesin the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids ofgreater than 100 nucleotides in length, equations for calculating T_(m)have been derived (see Sambrook et al., supra, 9.50-0.51). Forhybridization with shorter nucleic acids, i.e., oligonucleotides, theposition of mismatches becomes more important, and the length of theoligonucleotide determines its specificity (see Sambrook et al., supra,11.7-11.8).

Selectivity of hybridization exists when hybridization occurs which ismore selective than total lack of specificity. Typically, selectivehybridization will occur when there is at least about 55% homology overa stretch of at least about 14/25 nucleotides, preferably at least about65%, more preferably at least about 75%, and most preferably at leastabout 90%. See, Kanehisa, M. (1984), Nucleic Acids Res. 12:203-213,which is incorporated herein by reference. Stringent hybridizationconditions will typically include salt concentrations of less than about1 M, more usually less than about 500 mM and preferably less than about200 mM. Temperature conditions will typically be greater than 20 degreesCelsius, more usually greater than about 30 degrees Celsius andpreferably in excess of about 37 degrees Celsius. As other factors maysignificantly affect the stringency of hybridization, including, amongothers, base composition and size of the complementary strands, presenceof organic solvents and extent of base mismatching, the combination ofparameters is more important than the absolute measure of any one.

In a specific embodiment of the invention, polynucleotides of theinvention are detected by employing hybridization conditions comprisinga hybridization step in less than 500 mM salt and at least 37 degreesCelsius, and a washing step in 2×SSPE at least 63 degrees Celsius. In apreferred embodiment, the hybridization conditions comprise less than200 mM salt and at least 37 degrees Celsius for the hybridization step.In a more preferred embodiment, the hybridization conditions comprise2×SSPE and 63 degrees Celsius for both the hybridization and washingsteps.

In one embodiment, the length for a hybridizable nucleic acid is atleast about 10 nucleotides. Preferable a minimum length for ahybridizable nucleic acid is at least about 15 nucleotides; morepreferably at least about 20 nucleotides; and even more preferably thelength is at least 30 nucleotides. Furthermore, the skilled artisan willrecognize that the temperature and wash solution salt concentration maybe adjusted as necessary according to factors such as length of theprobe.

The term “probe” refers to a single-stranded nucleic acid molecule thatcan base pair with a complementary single stranded target nucleic acidto form a double-stranded molecule.

As used herein, the term “oligonucleotide” refers to a nucleic acid,generally of at least 18 nucleotides, that is hybridizable to a genomicDNA molecule, a cDNA molecule, a plasmid DNA or an mRNA molecule.Oligonucleotides can be labeled, e.g., with ³²P-nucleotides ornucleotides to which a label, such as biotin, has been covalentlyconjugated. A labeled oligonucleotide can be used as a probe to detectthe presence of a nucleic acid. Oligonucleotides (one or both of whichmay be labeled) can be used as PCR primers, either for cloning fulllength or a fragment of a nucleic acid, or to detect the presence of anucleic acid. An oligonucleotide can also be used to form a triple helixwith a DNA molecule. Generally, oligonucleotides are preparedsynthetically, preferably on a nucleic acid synthesizer. Accordingly,oligonucleotides can be prepared with non-naturally occurringphosphoester analog bonds, such as thioester bonds, etc.

A “primer” is an oligonucleotide that hybridizes to a target nucleicacid sequence to create a double stranded nucleic acid region that canserve as an initiation point for DNA synthesis under suitableconditions. Such primers may be used in a polymerase chain reaction.

“Polymerase chain reaction” is abbreviated PCR and means an in vitromethod for enzymatically amplifying specific nucleic acid sequences. PCRinvolves a repetitive series of temperature cycles with each cyclecomprising three stages: denaturation of the template nucleic acid toseparate the strands of the target molecule, annealing a single strandedPCR oligonucleotide primer to the template nucleic acid, and extensionof the annealed primer(s) by DNA polymerase. PCR provides a means todetect the presence of the target molecule and, under quantitative orsemi-quantitative conditions, to determine the relative amount of thattarget molecule within the starting pool of nucleic acids.

“Reverse transcription-polymerase chain reaction” is abbreviated RT-PCRand means an in vitro method for enzymatically producing a target cDNAmolecule or molecules from an RNA molecule or molecules, followed byenzymatic amplification of a specific nucleic acid sequence or sequenceswithin the target cDNA molecule or molecules as described above. RT-PCRalso provides a means to detect the presence of the target molecule and,under quantitative or semi quantitative conditions, to determine therelative amount of that target molecule within the starting pool ofnucleic acids.

A DNA “coding sequence” is a double-stranded DNA sequence that istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.“Suitable regulatory sequences” refer to nucleotide sequences locatedupstream (5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, polyadenylation recognition sequences, RNAprocessing site, effector binding site and stem-loop structure. Theboundaries of the coding sequence are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl)terminus. A coding sequence can include, but is not limited to,prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and evensynthetic DNA sequences. If the coding sequence is intended forexpression in a eukaryotic cell, a polyadenylation signal andtranscription termination sequence will usually be located 3′ to thecoding sequence.

“Open reading frame” is abbreviated ORF and means a length of nucleicacid sequence, either DNA, cDNA or RNA, that comprises a translationstart signal or initiation codon, such as an ATG or AUG, and atermination codon and can be potentially translated into a polypeptidesequence.

The term “head-to-head” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a head-to-head orientation when the 5′end of the coding strand of one polynucleotide is adjacent to the 5′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds away from the 5′ end ofthe other polynucleotide. The term “head-to-head” may be abbreviated(5′)-to-(5′) and may also be indicated by the symbols (←→) or(3′←5′5′→3′).

The term “tail-to-tail” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a tail-to-tail orientation when the 3′end of the coding strand of one polynucleotide is adjacent to the 3′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds toward the otherpolynucleotide. The term “tail-to-tail” may be abbreviated (3′)-to-(3′)and may also be indicated by the symbols (→←) or (5′→3′3′←5′).

The term “head-to-tail” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a head-to-tail orientation when the 5′end of the coding strand of one polynucleotide is adjacent to the 3′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds in the same directionas that of the other polynucleotide. The term “head-to-tail” may beabbreviated (5′)-to-(3′) and may also be indicated by the symbols (→→)or (5′→3′5′→3′).

The term “downstream” refers to a nucleotide sequence that is located 3′to reference nucleotide sequence. In particular, downstream nucleotidesequences generally relate to sequences that follow the starting pointof transcription. For example, the translation initiation codon of agene is located downstream of the start site of transcription.

The term “upstream” refers to a nucleotide sequence that is located 5′to reference nucleotide sequence. In particular, upstream nucleotidesequences generally relate to sequences that are located on the 5′ sideof a coding sequence or starting point of transcription. For example,most promoters are located upstream of the start site of transcription.

The terms “restriction endonuclease” and “restriction enzyme” refer toan enzyme that binds and cuts within a specific nucleotide sequencewithin double stranded DNA.

“Homologous recombination” refers to the insertion of a foreign DNAsequence into another DNA molecule, e.g., insertion of a vector in achromosome. Preferably, the vector targets a specific chromosomal sitefor homologous recombination. For specific homologous recombination, thevector will contain sufficiently long regions of homology to sequencesof the chromosome to allow complementary binding and incorporation ofthe vector into the chromosome. Longer regions of homology, and greaterdegrees of sequence similarity, may increase the efficiency ofhomologous recombination.

Several methods known in the art may be used to propagate apolynucleotide according to the invention. Once a suitable host systemand growth conditions are established, recombinant expression vectorscan be propagated and prepared in quantity. As described herein, theexpression vectors which can be used include, but are not limited to,the following vectors or their derivatives: human or animal viruses suchas vaccinia virus or adenovirus; insect viruses such as baculovirus;yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid andcosmid DNA vectors, to name but a few.

A “vector” is any means for the cloning of and/or transfer of a nucleicacid into a host cell. A vector may be a replicon to which another DNAsegment may be attached so as to bring about the replication of theattached segment. A “replicon′ is any genetic element (e.g., plasmid,phage, cosmid, chromosome, virus) that functions as an autonomous unitof DNA replication in vivo, i.e., capable of replication under its owncontrol. The term “vector” includes both viral and nonviral means forintroducing the nucleic acid into a cell in vitro, ex vivo or in vivo. Alarge number of vectors known in the art may be used to manipulatenucleic acids, incorporate response elements and promoters into genes,etc. Possible vectors include, for example, plasmids or modified virusesincluding, for example bacteriophages such as lambda derivatives, orplasmids such as pBR322 or pUC plasmid derivatives, or the Bluescriptvector. For example, the insertion of the DNA fragments corresponding toresponse elements and promoters into a suitable vector can beaccomplished by ligating the appropriate DNA fragments into a chosenvector that has complementary cohesive termini. Alternatively, the endsof the DNA molecules may be enzymatically modified or any site may beproduced by ligating nucleotide sequences (linkers) into the DNAtermini. Such vectors may be engineered to contain selectable markergenes that provide for the selection of cells that have incorporated themarker into the cellular genome. Such markers allow identificationand/or selection of host cells that incorporate and express the proteinsencoded by the marker.

Viral vectors, and particularly retroviral vectors, have been used in awide variety of gene delivery applications in cells, as well as livinganimal subjects. Viral vectors that can be used include but are notlimited to retrovirus, adeno-associated virus, pox, baculovirus,vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, andcaulimovirus vectors. Non-viral vectors include plasmids, liposomes,electrically charged lipids (cytofectins), DNA-protein complexes, andbiopolymers. In addition to a nucleic acid, a vector may also compriseone or more regulatory regions, and/or selectable markers useful inselecting, measuring, and monitoring nucleic acid transfer results(transfer to which tissues, duration of expression, etc.).

The term “plasmid” refers to an extra chromosomal element often carryinga gene that is not part of the central metabolism of the cell, andusually in the form of circular double-stranded DNA molecules. Suchelements may be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear, circular, orsupercoiled, of a single- or double-stranded DNA or RNA, derived fromany source, in which a number of nucleotide sequences have been joinedor recombined into a unique construction which is capable of introducinga promoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell.

A “cloning vector” is a “replicon”, which is a unit length of a nucleicacid, preferably DNA, that replicates sequentially and which comprisesan origin of replication, such as a plasmid, phage or cosmid, to whichanother nucleic acid segment may be attached so as to bring about thereplication of the attached segment. Cloning vectors may be capable ofreplication in one cell type and expression in another (“shuttlevector”).

Vectors may be introduced into the desired host cells by methods knownin the art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), particle bombardment, useof a gene gun, or a DNA vector transporter (see, e.g., Wu et al., 1992,J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem.263:14621-14624; and Hartmut et al., Canadian Patent Application No.2,012,311, filed Mar. 15, 1990).

A polynucleotide according to the invention can also be introduced invivo by lipofection. For the past decade, there has been increasing useof liposomes for encapsulation and transfection of nucleic acids invitro. Synthetic cationic lipids designed to limit the difficulties anddangers encountered with liposome-mediated transfection can be used toprepare liposomes for in vivo transfection of a gene encoding a marker(Felgner et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84: 7413; Mackey,et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85: 8027-8031; and Ulmer etal., 1993, Science 259: 1745-1748). The use of cationic lipids maypromote encapsulation of negatively charged nucleic acids, and alsopromote fusion with negatively charged cell membranes (Felgner andRingold, 1989. Science 337:387-388). Particularly useful lipid compoundsand compositions for transfer of nucleic acids are described inInternational Patent Publications WO95/18863 and WO96/17823, and in U.S.Pat. No. 5,459,127. The use of lipofection to introduce exogenous genesinto the specific organs in vivo has certain practical advantages.Molecular targeting of liposomes to specific cells represents one areaof benefit. It is clear that directing transfection to particular celltypes would be particularly preferred in a tissue with cellularheterogeneity, such as pancreas, liver, kidney, and the brain. Lipidsmay be chemically coupled to other molecules for the purpose oftargeting (Mackey, et al., 1988, supra). Targeted peptides, e.g.,hormones or neurotransmitters, and proteins such as antibodies, ornon-peptide molecules could be coupled to liposomes chemically.

Other molecules are also useful for facilitating transfection of anucleic acid in vivo, such as a cationic oligopeptide (e.g.,WO95/21931), peptides derived from DNA binding proteins (e.g., WO96/25508), or a cationic polymer (e.g., WO95/21931).

It is also possible to introduce a vector in vivo as a naked DNA plasmid(see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859).Receptor-mediated DNA delivery approaches can also be used (Curiel etal., 1992, Hum. Gene Ther. 3; 147-154; and Wu and Wu, 1987, J. Biol.Chem. 262:4429-4432).

The term “transfection” means the uptake of exogenous or heterologousRNA or DNA by a cell. A cell has been “transfected” by exogenous orheterologous RNA or DNA when such RNA or DNA has been introduced insidethe cell. A cell has been “transformed” by exogenous or heterologous RNAor DNA when the transfected RNA or DNA effects a phenotypic change. Thetransforming RNA or DNA can be integrated (covalently linked) intochromosomal DNA marking up the genome of the cell.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The term “genetic region” will refer to a region of a nucleic acidmolecule or a nucleotide sequence that comprises a gene encoding apolypeptide.

In addition, the recombinant vector comprising a polynucleotideaccording to the invention may include one or more origins forreplication in the cellular hosts in which their amplification or theirexpression is sought, markers or selectable markers.

The term “selectable marker” means an identifying factor, usually anantibiotic or chemical resistance gene, that is able to be selected forbased upon the marker gene's effect, i.e., resistance to an antibiotic,resistance to a herbicide, colorimetric markers, enzymes, fluorescentmarkers, and the like, wherein the effect is used to track theinheritance of a nucleic acid of interest and/or to identify a cell ororganism that has inherited the nucleic acid of interest. Examples ofselectable marker genes known and used in the art include: genesproviding resistance to ampicillin, streptomycin, gentamycin, kanamycin,hygromycin, bialaphos herbicide, sulfonamide, and the like; and genesthat are used as phenotypic markers, i.e., anthocyanin regulatory genes,isopentanyl transferase gene, and the like. Selectable marker genes mayalso be considered reporter genes.

The term “reporter gene” means a nucleic acid encoding an identifyingfactor that is able to be identified based upon the reporter gene'seffect, wherein the effect is used to track the inheritance of a nucleicacid of interest, to identify a cell or organism that has inherited thenucleic acid of interest, and/or to measure gene expression induction ortranscription. Examples of reporter genes known and used in the artinclude: luciferase (Luc), green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ),β-glucuronidase (Gus), and the like.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. Promotersthat cause a gene to be expressed in a specific cell type are commonlyreferred to as “cell-specific promoters” or “tissue-specific promoters”.Promoters that cause a gene to be expressed at a specific stage ofdevelopment or cell differentiation are commonly referred to as“developmentally-specific promoters” or “cell differentiation-specificpromoters”. Promoters that are induced and cause a gene to be expressedfollowing exposure or treatment of the cell with an agent, biologicalmolecule, chemical, ligand, light, or the like that induces the promoterare commonly referred to as “inducible promoters” or “regulatablepromoters”. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined, DNAfragments of different lengths may have identical promoter activity.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced (if the coding sequence contains introns) and translated intothe protein encoded by the coding sequence.

“Transcriptional and translational control sequences” are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences.

The term “response element” means one or more cis-acting DNA elementswhich confer responsiveness on a promoter mediated through interactionwith the DNA-binding domains of the first chimeric gene. This DNAelement may be either palindromic (perfect or imperfect) in its sequenceor composed of sequence motifs or half sites separated by a variablenumber of nucleotides. The half sites can be similar or identical andarranged as either direct or inverted repeats or as a single half siteor multimers of adjacent half sites in tandem. The response element maycomprise a minimal promoter isolated from different organisms dependingupon the nature of the cell or organism into which the response elementwill be incorporated. The DNA binding domain of the first hybrid proteinbinds, in the presence or absence of a ligand, to the DNA sequence of aresponse element to initiate or suppress transcription of downstreamgene(s) under the regulation of this response element. Examples of DNAsequences for response elements of the natural ecdysone receptorinclude: RRGG/TTCANTGAC/ACYY (see Cherbas L., et. al., (1991), GenesDev. 5:120-131); AGGTCAN_((n))AGGTCA, where N_((n)) can be one or morespacer nucleotides (see D'Avino P P., et. al., (1995), Mol. Cell.Endocrinol, 113: 1-9); and GGGTTGAATGAATTT (see Antoniewski C., et. al.,(1994), Mol. Cell Biol. 14: 4465-4474).

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from anucleic acid or polynucleotide. Expression may also refer to translationof mRNA into a protein or polypeptide.

The terms “cassette”, “expression cassette” and “gene expressioncassette” refer to a segment of DNA that can be inserted into a nucleicacid or polynucleotide at specific restriction sites or by homologousrecombination. The segment of DNA comprises a polynucleotide thatencodes a polypeptide of interest, and the cassette and restrictionsites are designed to ensure insertion of the cassette in the properreading frame for transcription and translation. “Transformationcassette” refers to a specific vector comprising a polynucleotide thatencodes a polypeptide of interest and having elements in addition to thepolynucleotide that facilitate transformation of a particular host cell.Cassettes, expression cassettes, gene expression cassettes andtransformation cassettes of the invention may also comprise elementsthat allow for enhanced expression of a polynucleotide encoding apolypeptide of interest in a host cell. These elements may include, butare not limited to: a promoter, a minimal promoter, an enhancer, aresponse element, a terminator sequence, a polyadenylation sequence, andthe like.

For purposes of this invention, the term “gene switch” refers to thecombination of a response element associated with a promoter, and anEcR-based gene expression system which, in the presence of one or moreligands, modulates the expression of a gene into which the responseelement and promoter are incorporated.

The terms “modulate” and “modulates” mean to induce, reduce or inhibitnucleic acid or gene expression, resulting in the respective induction,reduction or inhibition of protein or polypeptide production.

The plasmids or vectors according to the invention may further compriseat least one promoter suitable for driving expression of a gene in ahost cell. The term “expression vector” means a vector, plasmid orvehicle designed to enable the expression of an inserted nucleic acidsequence following transformation into the host. The cloned gene, i.e.,the inserted nucleic acid sequence, is usually placed under the controlof control elements such as a promoter, a minimal promoter, an enhancer,or the like. Initiation control regions or promoters, which are usefulto drive expression of a nucleic acid in the desired host cell arenumerous and familiar to those skilled in the art. Virtually anypromoter capable of driving these genes is suitable for the presentinvention including but not limited to: viral promoters, bacterialpromoters, animal promoters, mammalian promoters, synthetic promoters,constitutive promoters, tissue specific promoter, developmental specificpromoters, inducible promoters, light regulated promoters; CYC1, HIS3,GAL1, GAL4, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO,TPI, alkaline phosphatase promoters (useful for expression inSaccharomyces); AOX1 promoter (useful for expression in Pichia);b-lactamase, lac, ara, tet, trp, lP_(L), lP_(R), 17, tac, and trcpromoters (useful for expression in Escherichia coli); light regulated-,seed specific-, pollen specific-, ovary specific-, pathogenesis ordisease related-, cauliflower mosaic virus 35S, CMV 35S minimal, cassavavein mosaic virus (CsVMV), chlorophyll a/b binding protein, ribulose1,5-bisphosphate carboxylase, shoot-specific, root specific, chitinase,stress inducible, rice tungro bacilliform virus, plant super-promoter,potato leucine aminopeptidase, nitrate reductase, mannopine synthase,nopaline synthase, ubiquitin, zein protein, and anthocyanin promoters(useful for expression in plant cells); animal and mammalian promotersknown in the art include, but are not limited to, the SV40 early (SV40e)promoter region, the promoter contained in the 3′ long terminal repeat(LTR) of Rous sarcoma virus (RSV), the promoters of the E1A or majorlate promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus(CMV) early promoter, the herpes simplex virus (HSV) thymidine kinaseMK) promoter, a baculovirus IE1 promoter, an elongation factor 1 alpha(EF1) promoter, a phosphoglycerate kinase (PGK) promoter, a ubiquitin(Ubc) promoter, an albumin promoter, the regulatory sequences of themouse metallothionein-L promoter and transcriptional control regions,the ubiquitous promoters (HPRT, vimentin, α-actin, tubulin and thelike), the promoters of the intermediate filaments (desmin,neurofilaments, keratin, GFAP, and the like), the promoters oftherapeutic genes (of the MDR, CFFR or factor VIII type, and the like),pathogenesis or disease related-promoters, and promoters that exhibittissue specificity and have been utilized in transgenic animals, such asthe elastase I gene control region which is active in pancreatic acinarcells; insulin gene control region active in pancreatic beta cells,immunoglobulin gene control region active in lymphoid cells, mousemammary tumor virus control region active in testicular, breast,lymphoid and mast cells; albumin gene, Apo AI and Apo AII controlregions active in liver, alpha-fetoprotein gene control region active inliver, alpha 1-antitrypsin gene control region active in the liver,beta-globin gene control region active in myeloid cells, myelin basicprotein gene control region active in oligodendrocyte cells in thebrain, myosin light chain-2 gene control region active in skeletalmuscle, and gonadotropic releasing hormone gene control region active inthe hypothalamus, pyruvate kinase promoter, villin promoter, promoter ofthe fatty acid binding intestinal protein, promoter of the smooth musclecell a-actin, and the like. In addition, these expression sequences maybe modified by addition of enhancer or regulatory sequences and thelike.

Enhancers that may be used in embodiments of the invention include butare not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer,an elongation factor 1 (EF1) enhancer, yeast enhancers, viral geneenhancers, and the like.

Termination control regions, i;e., terminator or polyadenylationsequences, may also be derived from various genes native to thepreferred hosts. Optionally, a termination site may be unnecessary,however, it is most preferred if included. In a preferred embodiment ofthe invention, the termination control region may be comprise or bederived from a synthetic sequence, synthetic polyadenylation signal, anSV40 late polyadenylation signal, an SV40 polyadenylation signal, abovine growth hormone (BGH) polyadenylation signal, viral terminatorsequences, or the like.

The terms “3′ non-coding sequences” or “3′ untranslated region (UTR)”refer to DNA sequences located downstream (3′) of a coding sequence andmay comprise polyadenylation [poly(A)] recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor.

“Regulatory region” means a nucleic acid sequence that regulates theexpression of a second nucleic acid sequence. A regulatory region mayinclude sequences which are naturally responsible for expressing aparticular nucleic acid (a homologous region) or may include sequencesof a different origin that are responsible for expressing differentproteins or even synthetic proteins (a heterologous region). Inparticular, the sequences can be sequences of prokaryotic, eukaryotic,or viral genes or derived sequences that stimulate or represstranscription of a gene in a specific or non-specific manner and in aninducible or non-inducible manner. Regulatory regions include origins ofreplication, RNA splice sites, promoters, enhancers, transcriptionaltermination sequences, and signal sequences which direct the polypeptideinto the secretory pathways of the target cell.

A regulatory region from a “heterologous source” is a regulatory regionthat is not naturally associated with the expressed nucleic acid.Included among the heterologous regulatory regions are regulatoryregions from a different species, regulatory regions from a differentgene, hybrid regulatory sequences, and regulatory sequences which do notoccur in nature, but which are designed by one having ordinary skill inthe art.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene. The complementarity of anantisense RNA may be with any part of the specific gene transcript,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, or thecoding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA,or other RNA that is not translated yet has an effect on cellularprocesses.

A “polypeptide” is a polymeric compound comprised of covalently linkedamino acid residues. Amino acids have the following general structure:

Amino acids are classified into seven groups on the basis of the sidechain R: (1) aliphatic side chains, (2) side chains containing ahydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) sidechains containing an acidic or amide group, (5) side chains containing abasic group, (6) side chains containing an aromatic ring, and (7)proline, an imino acid in which the side chain is fused to the aminogroup. A polypeptide of the invention preferably comprises at leastabout 14 amino acids.

A “protein” is a polypeptide that performs a structural or functionalrole in a living cell.

An “isolated polypeptide” or “isolated protein” is a polypeptide orprotein that is substantially free of those compounds that are normallyassociated therewith in its natural state (e.g., other proteins orpolypeptides, nucleic acids, carbohydrates, lipids). “Isolated” is notmeant to exclude artificial or synthetic mixtures with other compounds,or the presence of impurities which do not interfere with biologicalactivity, and which may be present, for example, due to incompletepurification, addition of stabilizers, or compounding into apharmaceutically acceptable preparation.

A “fragment” of a polypeptide according to the invention will beunderstood to mean a polypeptide whose amino acid sequence is shorterthan that of the reference polypeptide and which comprises, over theentire portion with these reference polypeptides, an identical aminoacid sequence. Such fragments may, where appropriate, be included in alarger polypeptide of which they are a part. Such fragments of apolypeptide according to the invention may have a length of at least2-300 amino acids.

A “heterologous protein” refers to a protein not naturally produced inthe cell.

A “mature protein” refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or propeptides present in theprimary translation product have been removed. “Precursor” proteinrefers to the primary product of translation of RNA; i.e., with pre- andpropeptides still present. Pre- and propeptides may be but are notlimited to intracellular localization signals.

The term “signal peptide” refers to an amino terminal polypeptidepreceding the secreted mature protein. The signal peptide is cleavedfrom and is therefore not present in the mature protein. Signal peptideshave the function of directing and translocating secreted proteinsacross cell membranes. Signal peptide is also referred to as signalprotein.

A “signal sequence” is included at the beginning of the coding sequenceof a protein to be expressed on the surface of a cell. This sequenceencodes a signal peptide, N-terminal to the mature polypeptide, thatdirects the host cell to translocate the polypeptide. The term“translocation signal sequence” is used herein to refer to this sort ofsignal sequence. Translocation signal sequences can be found associatedwith a variety of proteins native to eukaryotes and prokaryotes, and areoften functional in both types of organisms.

The term “homology” refers to the percent of identity between twopolynucleotide or two polypeptide moieties. The correspondence betweenthe sequence from one moiety to another can be determined by techniquesknown to the art. For example, homology can be determined by a directcomparison of the sequence information between two polypeptide moleculesby aligning the sequence information and using readily availablecomputer programs. Alternatively, homology can be determined byhybridization of polynucleotides under conditions that form stableduplexes between homologous regions, followed by digestion withsingle-stranded-specific nuclease(s) and size determination of thedigested fragments.

As used herein, the term “homologous” in all its grammatical forms andspelling variations refers to the relationship between proteins thatpossess a “common evolutionary origin,” including proteins fromsuperfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al., 1987, Cell 50: 667.). Such proteins (and their encoding genes)have sequence homology, as reflected by their high degree of sequencesimilarity. However, in common usage and in the instant application, theterm “homologous,” when modified with an adverb such as “highly,” mayrefer to sequence similarity and not a common evolutionary origin.

Accordingly, the term “sequence similarity” in all its gramatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that may or may not share a commonevolutionary origin (see Reeck et al., 1987, Cell 50: 667).

In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 50%(preferably at least about 75%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Sambrook et al., 1989, supra.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the protein encoded by the DNA sequence. “Substantially similar” alsorefers to nucleic acid fragments wherein changes in one or morenucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotide bases that donot substantially affect the functional properties of the resultingtranscript. It is therefore understood that the invention encompassesmore than the specific exemplary sequences. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts.

Moreover, the skilled artisan recognizes that substantially similarsequences encompassed by this invention are also defined by theirability to hybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65°C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS), withthe sequences exemplified herein. Substantially similar nucleic acidfragments of the instant invention are those nucleic acid fragmentswhose DNA sequences are at least 70% identical to the DNA sequence ofthe nucleic acid fragments reported herein. Preferred substantiallynucleic acid fragments of the instant invention are those nucleic acidfragments whose DNA sequences are at least 80% identical to the DNAsequence of the nucleic acid fragments reported herein. More preferrednucleic acid fragments are at least 90% identical to the DNA sequence ofthe nucleic acid fragments reported herein. Even more preferred arenucleic acid fragments that are at least 95% identical to the DNAsequence of the nucleic acid fragments reported herein.

Two amino acid sequences are “substantially homologous” or“substantially similar” when greater than about 40% of the amino acidsare identical, or greater than 60% are similar (finctionally identical).Preferably, the similar or homologous sequences are identified byalignment using, for example, the GCG (Genetics Computer Group, ProgramManual for the GCG Package, Version 7, Madison, Wis.) pileup program.

The term “corresponding to” is used herein to refer to similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. A nucleic acid or amino acid sequence alignment may includespaces. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

A “substantial portion” of an amino acid or nucleotide sequencecomprises enough of the amino acid sequence of a polypeptide or thenucleotide sequence of a gene to putatively identify that polypeptide orgene, either by manual evaluation of the sequence by one skilled in theart, or by computer-automated sequence comparison and identificationusing algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul, S. F., et al., (1993), J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identify and/or isolate a nucleic acid fragment comprisingthe sequence.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, New York (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, New York (1993); Computer Analysis of Sequence Data,Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NewJersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G.,ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M.and Devereux, J., eds.) Stockton Press, New York (1991). Preferredmethods to determine identity are designed to give the best matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequencesmay be performed using the Clustal method of alignment (Higgins andSharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method may be selected: KTUPLE 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include but is not limited to the GCG suite of programs (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.),BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215: 403-410(1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715USA). Within the context of this application it will be understood thatwhere sequence analysis software is used for analysis, that the resultsof the analysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters which originally load with thesoftware when first initialized.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments that are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-tablished procedures, orautomated chemical synthesis can be performed using one of a number ofcommercially available machines. Accordingly, the genes can be tailoredfor optimal gene expression based on optimization of nucleotide sequenceto reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

Polynucleotides Encoding Leafhopper Ecdysone Receptor Polypeptides

The present invention provides novel polynucleotides encoding aleafhopper ecdysone receptor polypeptide of the invention, including afull-length leafhopper ecdysone receptor protein, and any leafhopperecdysone receptor-specific fragments thereof.

In accordance with specific embodiments of the present invention,nucleic acid sequences encoding portions of a novel ecdysone receptorpolypeptide have been elucidated and characterized. Specifically,polynucleotides encoding a homopteran ecdysone receptor from greenleafhopper NcEcR) have been characterized. The full-length encodingsequence has been determined and is presented herein as SEQ ID NO: 1. Inaddition, domains within this polynucleotide encoding the full-lengthNcEcR polypeptide have been defined and are presented herein asdescribed in Table 1. TABLE 1 Nucleotide and amino acid sequencescorresponding to various domains and helices of leafhopper ecdysonereceptor (“NcEcR”). Full Length NcEcR or NcEcR Nucleotides of AminoAcids of Domains SEQ ID NO: 1 SEQ ID NO: 2 A/BCDE (Full length)  1-1109 1-370 A/B 1-15 1-5 C 16-213  6-71 D 214-447   72-149 E 448-1109 150-370CDE  16-1109  6-370 DE 214-1109  72-370 Helices 1-12 418-1109 140-370

Thus, a first subject of the invention relates to an isolatedpolynucleotide encoding a novel ecdysone receptor polypeptide. Morespecifically, the invention relates to an isolated polynucleotideencoding a leafhopper ecdysone receptor polypeptide. In a specificembodiment, the isolated polynucleotide comprises a nucleic acidsequence selected from the group consisting of SEQ ID NO: 1, nucleotides1-15 of SEQ ID NO: 1, nucleotides 16-213 of SEQ ID NO: 1, nucleotides214-447 of SEQ ID NO: 1, nucleotides 448-1109 of SEQ ID NO: 1,nucleotides 16-1109 of SEQ ID NO: 1, nucleotides 214-1109 of SEQ ID NO:1, and nucleotides 418-1109 of SEQ ID NO: 1. In another specificembodiment, the isolated polynucleotide comprises a nucleic acidsequence as depicted in SEQ ID NO: 1. In another specific embodiment,the isolated polynucleotide further comprises a region permittingexpression of the polypeptide in a host cell.

The present invention also relates to an isolated polynucleotideencoding a polypeptide comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 2, amino acids 1-5 of SEQ ID NO: 2,amino acids 6-71 of SEQ ID NO: 2, amino acids 72-149 of SEQ ID NO: 2,amino acids 150-370 of SEQ ID NO: 2, amino acids 6-370 of SEQ ID NO: 2,amino acids 72-370 of SEQ ID NO: 2, and amino acids 140-370 of SEQ IDNO: 2. In a specific embodiment, the isolated polynucleotide encodes aleafhopper ecdysone receptor polypeptide comprising an amino acidsequence as depicted in SEQ ID NO: 2.

The present invention provides novel isolated polynucleotides encodingleafhopper ecdysone receptor polypeptides. Having elucidated thesequence and structure of this ecdysone receptor, an isolatedpolynucleotide encoding a leafhopper receptor polypeptide comprising aligand-binding domain may be used individually or in combination toscreen for new ligands that bind this ligand binding domain. Thus, forexample, an ecdysone receptor polypeptide according to the invention maybe used to control expression of reporter genes for which sensitiveassays exist. The ligand binding domain may serve as a reagent forscreening new molecules, useful as either agonists or antagonists of theleafhopper ecdysone receptor. Either new classes of molecules may bescreened, or selected modifications from known ligands may be used.These new ligands find use as highly specific and highly active,naturally occurring pesticides. Thus, the present invention provides forscreening for new ligand molecules.

The polynucleotides of the present invention also provide probes forscreening for homologous nucleic acid sequences, both in Nephotetix andother genetic sources. This screening allows isolation of homologousgenes from both vertebrates and invertebrates.

Accordingly, any leafhopper cell potentially can serve as the nucleicacid source for the molecular cloning of a leafhopper ecdysone receptorpolynucleotide. The polynucleotide may be obtained by standardprocedures known in the art from cloned DNA (e.g., a DNA “library”), andpreferably is obtained from a cDNA library prepared from tissues withhigh level expression of the protein, by chemical synthesis, by cDNAcloning, or by the cloning of genomic DNA, or fragments thereof,purified from the desired cell (See, for example, Sambrook et al., 1989,supra; Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRLPress, Ltd., Oxford, U.K. Vol. I, II). Clones derived from genomic DNAmay contain regulatory and intron DNA regions in addition to codingregions; clones derived from cDNA will not contain intron sequences.Whatever the source, the polynucleotide should be molecularly clonedinto a suitable vector for propagation of the polynucleotide.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired leafhopper ecdysone receptorpolynucleotide may be accomplished in a number of ways. For example, DNAfragments may be screened by nucleic acid hybridization to a labeledprobe (Benton and Davis, 1977, Science 196: 180; Grunstein and Hogness,1975, Proc. Natl. Acad. Sci. U.S.A. 72: 3961). Those DNA fragments withsubstantial homology to the probe will hybridize. As noted above, thegreater the degree of homology, the more stringent hybridizationconditions can be used.

Further selection can be carried out on the basis of the properties ofthe polynucleotide, e.g., if the polynucleotide encodes a polypeptidehaving the isoelectric, electrophoretic, amino acid composition, orpartial amino acid sequence of the leafhopper ecdysone receptorpolypeptide as disclosed herein. Thus, the presence of thepolynucleotide may be detected by assays based on the physical,chemical, or immunological properties of its expressed product. Forexample, cDNA clones, or DNA clones which hybrid-select the propermRNAs, can be selected which produce a polypeptide that, e.g., hassimilar or identical electrophoretic migration, isoelectric focusing ornon-equilibrium pH gel electrophoresis behavior, proteolytic digestionmaps, or antigenic properties as known for a leafhopper ecdysonereceptor polypeptide. In a specific embodiment, the expressedpolypeptide is recognized by a polyclonal antibody that is generatedagainst an epitope specific for a leafhopper ecdysone receptorpolypeptide.

Due to the degeneracy of nucleotide coding sequences, otherpolynucleotides that encode substantially the same amino acid sequenceas a leafhopper ecdysone receptor polynucleotide disclosed herein,including an amino acid sequence that contains a single amino acidvariant, may be used in the practice of the present invention. Theseinclude but are not limited to allelic genes, homologous genes fromother species, and nucleotide sequences comprising all or portions ofleafhopper ecdysone receptor polynucleotides that are altered by thesubstitution of different codons that encode the same amino acid residuewithin the sequence, thus producing a silent change. Likewise, theleafhopper ecdysone receptor derivatives of the invention include, butare not limited to, those comprising, as a primary amino acid sequence,all or part of the amino acid sequence of a leafhopper ecdysone receptorpolypeptide including altered sequences in which functionally equivalentamino acid residues are substituted for residues within the sequenceresulting in a conservative amino acid substitution. For example, one ormore amino acid residues within the sequence can be substituted byanother amino acid of a similar polarity, which acts as a functionalequivalent, resulting in a silent alteration. Substitutes for an aminoacid within the sequence may be selected from other members of the classto which the amino acid belongs. For example, the nonpolar hydrophobic)amino acids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. Amino acids containingaromatic ring structures are phenylalanine, tryptophan, and tyrosine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid Such alterations can be produced by various methods knownin the art (see Sambrook et al., 1989, infra) and are not expected toaffect apparent molecular weight as determined by polyacrylamide gelelectrophoresis, or isoelectric point.

The present invention also relates to an isolated leafhopper ecdysonereceptor polypeptide encoded by a polynucleotide according to theinvention.

Leafhopper Ecdysone Receptor Polypeptides

The present invention provides novel isolated leafhopper ecdysonereceptor polypeptides, including a full-length leafhopper ecdysonereceptor protein, and any leafhopper ecdysone receptor-specificpolypeptide fragments thereof.

Thus, the invention relates to an isolated ecdysone receptorpolypeptide. More specifically, the invention relates to an isolatedleafhopper ecdysone receptor polypeptide. In a specific embodiment, theisolated ecdysone receptor polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, amino acids 1-5 ofSEQ ID NO: 2, amino acids 6-71 of SEQ ID NO: 2, amino acids 72-149 ofSEQ ID NO: 2, amino acids 150-370 of SEQ ID NO: 2, amino acids 6-370 ofSEQ ID NO: 2, amino acids 72-370 of SEQ ID NO: 2, and amino acids140-370 of SEQ ID NO: 2. In another specific embodiment, the isolatedecdysone receptor polypeptide comprises an amino acid sequence asdepicted in SEQ ID NO: 2.

In another specific embodiment, the isolated ecdysone receptorpolypeptide is encoded by a polynucleotide comprising a nucleic acidsequence selected from the group consisting of SEQ ID NO: 1, nucleotides1-15 of SEQ ID NO: 1, nucleotides 16-213 of SEQ ID NO: 1, nucleotides214-447 of SEQ ID NO: 1, nucleotides 448-1109 of SEQ ID NO: 1,nucleotides 16-1109 of SEQ ID NO: 1, nucleotides 214-1109 of SEQ ID NO:1, and nucleotides 418-1109 of SEQ ID NO: 1. In another specificembodiment, the isolated ecdysone receptor polypeptide is encoded by apolynucleotide comprising a nucleic acid sequence as depicted in SEQ IDNO: 1.

One of skill in the art is able to produce other polynucleotides toencode the polypeptides of the invention, by making use of the presentinvention and the degeneracy or non-universality of the genetic code asdescribed herein.

Additional embodiments of the present invention include an ecdysonereceptor polypeptide according to the invention, wherein the ecdysonereceptor polypeptide is substantially free of naturally associated cellcomponents. Such polypeptides will typically be either full-lengthproteins, functional fragments, or fusion proteins comprising segmentsfrom an ecdysone receptor polypeptide of the present invention fused toa heterologous, or normally non-contiguous, protein domain. Preferably,the ecdysone receptor polypeptide comprises a transactivation domain, aDNA binding domain, a ligand binding domain, a hinge region, or aheterodimerization domain. More preferably, the ecdysone receptorpolypeptide comprises a ligand binding domain that is capable of bindingto a ligand selected from the group consisting of a steroid ligand and anon-steroid ligand. As desired, the ecdysone receptor polypeptide may befused to a second polypeptide to generate a hybrid polypeptide.Preferably, the second polypeptide is a heterologous polypeptide fromthe steroid hormone nuclear receptor superfamily.

Besides substantially full-length polypeptides, the present inventionprovides for biologically active fragments of the polypeptides.Significant biological activities include transactivation activity,ligand binding, DNA binding, heterodimerization activity, immunologicalactivity and other biological activities characteristic of steroidreceptor superfamily members. Immunological activities include bothimmunogenic function in a target immune system as well as sharing ofimmunological epitopes for binding, serving as either a competitor orsubstitute antigen for an ecdysone receptor epitope.

For example, transactivation, ligand binding, or DNA-binding domains maybe “swapped” between different new fusion polypeptides or fragments.Thus, novel hybrid polypeptides exhibiting new combinations ofspecificities result from the functional linkage of transactivation,ligand-binding specificities, or DNA-binding domains. This is extremelyuseful in the design of inducible expression systems.

For immunological purposes, immunogens may be produced that tandemlyrepeat polypeptide segments, thereby producing highly antigenicproteins. Alternatively, such polypeptides will serve as highlyefficient competitors for specific binding. Production of antibodies toNcEcR is described below.

The present invention also provides for other polypeptides comprisingfragments of NcEcR. Thus, fusion polypeptides between the NcEcR segmentsand other homologous or heterologous proteins are provided. Homologouspolypeptides may be fusions between different steroid receptorsuperfamily members, resulting in, for instance, a hybrid proteinexhibiting ligand specificity of one member and DNA-binding specificityof another. Likewise, heterologous fusions may be constructed whichwould exhibit a combination of properties or activities of thederivative proteins. Typical examples are fusions of a reporterpolypeptide, e.g., luciferase, with another domain of a receptor, e.g.,a DNA-binding domain, so that the presence or location of a desiredligand may be easily determined. See, e.g., Dull et al., U.S. Pat. No.4,859,609, which is hereby incorporated herein by reference. Othertypical gene fusion partners include “zinc finger” segment swappingbetween DNA-binding proteins, bacterial beta-galactosidase, trpE ProteinA, beta-lactamase, alpha amylase, alcohol dehydrogenase and yeast alphamating factor. See, e.g., Godowski et al. (1988), Science 241: 812-816.

Thus, the present invention also provides an isolated polypeptideselected from the group consisting of a) an isolated polypeptidecomprising a transactivation domain, a DNA-binding domain, and aleafhopper ecdysone receptor ligand binding domain; b) an isolatedpolypeptide comprising a DNA-binding domain and a leafhopper ecdysonereceptor ligand binding domain; and c) an isolated polypeptidecomprising a transactivation domain and a leafhopper ecdysone receptorligand binding domain. Preferably, the leafhopper ecdysone receptorligand binding domain comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2, amino acids 150-370 of SEQ ID NO: 2,amino acids 6-370 of SEQ ID NO: 2, amino acids 72-370 of SEQ ID NO: 2,and amino acids 140-370 of SEQ ID NO: 2. In another preferredembodiment, the leafhopper ecdysone receptor ligand binding domain isencoded by a polynucleotide comprising comprises a nucleic acid sequenceselected from the group consisting of SEQ ID NO: 1, nucleotides 448-1109of SEQ ID NO: 1, nucleotides 16-1109 of SEQ ID NO: 1, nucleotides214-1109 of SEQ ID NO: 1, and nucleotides 418-1109 of SEQ ID NO: 1.

The present invention also provides an isolated hybrid polypeptideselected from the group consisting of a) an isolated hybrid polypeptidecomprising a transactivation domain, a DNA-binding domain, and aleafhopper ecdysone receptor ligand binding domain; b) an isolatedhybrid polypeptide comprising a DNA-binding domain and a leafhopperecdysone receptor ligand binding domain; and c) an isolated hybridpolypeptide comprising a transactivation domain and a leafhopperecdysone receptor ligand binding domain. Preferably, the leafhopperecdysone receptor ligand binding domain comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, amino acids 150-370of SEQ ID NO: 2, amino acids 6-370 of SEQ ID NO: 2, amino acids 72-370of SEQ ID NO: 2, and amino acids 140-370 of SEQ ID NO: 2. In anotherpreferred embodiment, the leafhopper ecdysone receptor ligand bindingdomain is encoded by a polynucleotide comprises a nucleic acid sequenceselected from the group consisting of SEQ ID NO: 1, nucleotides 448-1109of SEQ ID NO: 1, nucleotides 16-1109 of SEQ ID NO: 1, nucleotides214-1109 of SEQ ID NO: 1, and nucleotides 418-1109 of SEQ ID NO: 1.

The present invention also relates to compositions comprising anisolated polypeptide according to the invention.

Compositions

The present invention also relates to compositions comprising theisolated polynucleotides or polypeptides according to the invention.Such compositions may comprise a leafhopper ecdysone receptorpolypeptide or a polynucleotide encoding a leafhopper ecdysone receptorpolypeptide, as defined above, and an acceptable carrier or vehicle. Thecompositions of the invention are particularly suitable for formulationof biological material for use in a gene expression modulation system ora ligand-screening assay according to the invention. Thus, in apreferred embodiment, the composition comprises a polynucleotideencoding a leafhopper ecdysone receptor polypeptide. In anotherpreferred embodiment, the composition comprises a leafhopper ecdysonereceptor polypeptide according to the invention.

The phrase “acceptable” refers to molecular entities and compositionsthat are physiologically tolerable to the cell or organism whenadministered. The term “carrier” refers to a diluent, adjuvant,excipient, or vehicle with which the composition is administered. Suchcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Examples ofacceptable carriers are saline, buffered saline, isotonic saline (e.g.,monosodium or disodium phosphate, sodium, potassium, calcium ormagnesium chloride, or mixtures of such salts), Ringer's solution,dextrose, water, sterile water, glycerol, ethanol, and combinationsthereof. 1,3-butanediol and sterile fixed oils are conveniently employedas solvents or suspending media. Any bland fixed oil can be employedincluding synthetic mono- or di-glycerides. Fatty acids such as oleicacid also find use in the preparation of injectables. Water or aqueoussolution saline solutions and aqueous dextrose and glycerol solutionsare preferably employed as carriers, particularly for injectablesolutions. Suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin. Pharmaceuticalcompositions of the invention may be formulated for the purpose oftopical, oral, parenteral, intranasal, intravenous, intramuscular,subcutaneous, intraocular, and the like, administration.

Preferably, the compositions comprise an acceptable vehicle for aninjectable formulation. This vehicle can be, in particular, a sterile,isotonic saline solution (monosodium or disodium phosphate, sodium,potassium, calcium or magnesium chloride, and the like, or mixtures ofsuch salts), or dry, in particular lyophilized, compositions which, onaddition, as appropriate, of sterilized water or of physiologicalsaline, enable injectable solutions to be formed The preferred sterileinjectable preparations can be a solution or suspension in a nontoxicparenterally acceptable solvent or diluent.

In yet another embodiment, a composition comprising a leafhopperecdysone receptor polypeptide, or polynucleotide encoding thepolypeptide, can be delivered in a controlled release system. Forexample, the polynucleotide or polypeptide may be administered usingintravenous infusion, an implantable osmotic pump, a transdermal patch,liposomes, or other modes of administration. Other controlled releasesystems are discussed in the review by Langer [Science 249: 1527-1533(1990)].

Expression of Leafhopper Ecdysone Receptor Polypeptides

With the sequence of the receptor polypeptides and the polynucleotidesencoding them, large quantities of leafhopper ecdysone receptorpolypeptides may be prepared. By the appropriate expression of vectorsin cells, high efficiency production may be achieved. Thereafter,standard purification methods may be used, such as ammonium sulfateprecipitations, column chromatography, electrophoresis, centrifugation,crystallization and others. See various volumes of Methods in Enzymologyfor techniques typically used for protein purification. Alternatively,in some embodiments high efficiency of production is unnecessary, butthe presence of a known inducing protein within a carefully engineeredexpression system is quite valuable. For instance, a combination of: (1)a ligand-responsive enhancer or response element operably linked to (2)a desired gene sequence with (3) the corresponding leafhopper ecdysonereceptor polypeptide together in an expression system provides aspecifically inducible expression system. Typically, the expressionsystem will be a cell, but an in vitro expression system may also beconstructed.

A polynucleotide encoding a leafhopper ecdysone receptor, or fragment,derivative or analog thereof, or a functionally active derivative,including a chimeric protein, thereof, can be inserted into anappropriate expression vector, i.e., a vector which comprises thenecessary elements for the transcription and translation of the insertedprotein-coding sequence. Thus, the polynucleotide of the invention isoperationally linked with a transcriptional control sequence in anexpression vector of the invention. Both cDNA and genomic sequences canbe cloned and expressed under control of such regulatory sequences. Anexpression vector also preferably includes a replication origin.

The isolated polynucleotides of the invention may be inserted into anyappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Examples of vectors include, but arenot limited to, Escherichia coli, bacteriophages such as lambdaderivatives, or plasmids such as pBR322 derivatives or pUC plasmidderivatives, e.g. pGEX vectors, pmal-c, pFLAG, etc. The insertion into acloning vector can, for example, be accomplished by ligating thepolynucleotide into a cloning vector that has complementary cohesivetermini. However, if the complementary restriction sites used tofragment the polynucleotide are not present in the cloning vector, theends of the polynucleotide molecules may be enzymatically modified.Alternatively, any site desired may be produced by ligating nucleotidesequences (linkers) onto the DNA termini; these ligated linkers maycomprise specific chemically synthesized oligonucleotides encodingrestriction endonuclease recognition sequences. Preferably, the clonedgene is contained on a shuttle vector plasmid, which provides forexpansion in a cloning cell, e.g., E. coli, and purification forsubsequent insertion into an appropriate expression cell line, if suchis desired. For example, a shuttle vector, which is a vector that canreplicate in more than one type of organism, can be prepared forreplication in both E. coli and Saccharomyces cerevisiae by linkingsequences from an E. coli plasmid with sequences form the yeast 2μplasmid.

In addition, the present invention relates to an expression vectorcomprising a polynucleotide according the invention, operatively linkedto a transcription regulatory element. Preferably, the polynucleotide isoperatively linked with an expression control sequence permittingexpression of the nuclear receptor ligand binding domain in anexpression competent host cell. The expression control sequence maycomprise a promoter that is functional in the host cell in whichexpression is desired. The vector may be a plasmid DNA molecule or aviral vector. Preferred viral vectors include retrovirus, adenovirus,adeno-associated virus, herpes virus, and vaccinia virus. The inventionfurther relates to a replication defective recombinant virus comprisingin its genome, a polynucleotide according to the invention. Thus, thepresent invention also relates to an isolated host cell comprising suchan expression vector, wherein the transcription regulatory element isoperative in the host cell.

The desired genes will be inserted into any of a wide selection ofexpression vectors. The selection of an appropriate vector and cell linedepends upon the constraints of the desired product. Typical expressionvectors are described in Sambrook et al. (1989). Suitable cell lines maybe selected from a depository, such as the ATCC. See, ATCC Catalogue ofCell Lines and Hybridomas (6th ed.) (1988); ATCC Cell Lines, Viruses,and Antisera, each of which is hereby incorporated herein by reference.The vectors are introduced to the desired cells by standardtransformation or transfection procedures as described, for instance, inSambrook et al. (1989).

Fusion proteins will typically be made by either recombinant nucleicacid methods or by synthetic polypeptide methods. Techniques for nucleicacid manipulation are described generally, for example, in Sambrook etal. (1989), Molecular Cloning: A Laboratory Manual (2d ed.), Vols. 1-3,Cold Spring Harbor Laboratory, which are incorporated herein byreference. Techniques for synthesis of polypeptides are described, forexample, in Merrifield, J. Amer. Chem. Soc. 85: 2149-2156 (1963).

The nucleotide sequences used to produce fusion proteins of the presentinvention may be derived from natural or synthetic sequences. Manynatural gene sequences are obtainable from various cDNA or from genomiclibraries using appropriate probes. See, GenBank™, National Institutesof Health. Typical probes for leafhopper ecdysone receptors may beselected from the sequences of Table 1 in accordance with standardprocedures. Suitable synthetic DNA fragments may be prepared by thephosphoramidite method described by Beaucage and Carruthers, Tetra.Letts. 22: 1859-1862 (1981). A double stranded fragment may then beobtained either by synthesizing the complementary strand and annealingthe strand together under appropriate conditions or by adding thecomplementary strand using DNA polymerase with an appropriate primersequence.

The natural or synthetic polynucleotide fragments encoding a desiredleafhopper ecdysone receptor polypeptide fragment will be incorporatedinto nucleic acid constructs capable of introduction to and expressionin an in vitro cell culture. Usually the nucleic acid constructs will besuitable for replication in a unicellular host, such as yeast orbacteria, but may also be intended for introduction to, with and withoutand integration within the genome, cultured mammalian or plant or othereukaryotic cell lines. Nucleic acid constructs prepared for introductioninto bacteria or yeast will typically include a replication systemrecognized by the host, the intended DNA fragment encoding the desiredreceptor polypeptide, transcription and translational initiationregulatory sequences operably linked to the polypeptide encoding segmentand transcriptional and translational termination regulatory sequencesoperably linked to the polypeptide encoding segment. The transcriptionalregulatory sequences will typically include a heterologous enhancer,response element, or promoter which is recognized by the host. Theselection of an appropriate promoter will depend upon the host, butpromoters such as the trp, lac and phage promoters, tRNA promoters andglycolytic enzyme promoters are known. See, Sambrook et al. (1989).Conveniently available expression vectors which include the replicationsystem and transcriptional and translational regulatory sequencestogether with the insertion site for the steroid receptor DNA sequencemay be employed. Examples of workable combinations of cell lines andexpression vectors are described in Sambrook et al. (1989); see also,Metzger et al. (1988), Nature 334: 31-36.

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity. Aspreviously explained, the expression vectors which can be used include,but are not limited to, the following vectors or their derivatives:human or animal viruses such as vaccinia virus or adenovirus; insectviruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g.,lambda), and plasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Different host cells havecharacteristic and specific mechanisms for the translational andpost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to ensure the desiredmodification and processing of the foreign protein expressed. Expressionin yeast can produce a biologically active product. Expression ineukaryotic cells can increase the likelihood of “native” folding.Moreover, expression in mammalian cells can provide a tool forreconstituting, or constituting, leafhopper ecdysone receptor activity.Furthermore, different vector/host expression systems may affectprocessing reactions, such as proteolytic cleavages, to a differentextent.

Vectors are introduced into the desired host cells by methods known inthe art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAB dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), particle bombardment, useof a gene gun, or a DNA vector transporter (see, e.g., Wu et al., 1992,J. Biol. Chem. 267: 963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311,filed Mar. 15, 1990).

Soluble forms of the protein can be obtained by collecting culturefluid, or solubilizing inclusion bodies, e.g., by treatment withdetergent, and if desired sonication or other mechanical processes, asdescribed above. The solubilized or soluble protein can be isolatedusing various techniques, such as polyacrylamide gel electrophoresis(PAGE), isoelectric focusing, 2-dimensional gel electrophoresis,chromatography (e.g., ion exchange, affinity, immunoaffinity, and sizingcolumn chromatography), centrifugation, differential solubility,immunoprecipitation, or by any other standard technique for thepurification of proteins.

Vectors and Gene Expression Cassettes Comprising a Leafhopper EcdysoneReceptor Polynucleotide

Thus, the present invention also relates to a vector comprising apolynucleotide encoding a leafhopper ecdysone receptor polypeptideaccording to the invention. The present invention also provides a geneexpression cassette comprising a polynucleotide encoding a leafhopperecdysone receptor polypeptide according to the invention. Thepolynucleotides of the invention, where appropriate incorporated invectors or gene expression cassettes, and the compositions comprisingthem, are useful for regulating gene expression in an ecdysonereceptor-based gene expression system. They may be used for the transferand expression of genes in vitro or in vivo in any type of cell ortissue. The transformation can, moreover, be targeted (transfer to aparticular tissue can, in particular, be determined by the choice of avector, and expression by the choice of a particular promoter). Thepolynucleotides and vectors of the invention are advantageously used forthe production in vivo and intracellularly, of polypeptides of interest.

The polynucleotides encoding the leafhopper ecdysone receptorpolypeptides of the invention will typically be used in a plasmidvector. Preferably, an expression control sequence is operably linked tothe leafhopper ecdysone receptor polynucleotide coding sequence forexpression of the leafhopper ecdysone receptor polypeptide. Theexpression control sequence may be any enhancer, response element, orpromoter system in vectors capable of transforming or transfecting ahost cell. Once the vector has been incorporated into the appropriatehost, the host, depending on the use, will be maintained underconditions suitable for high-level expression of the polynucleotides.

Polynucleotides will normally be expressed in hosts after the sequenceshave been operably linked to (i.e., positioned to ensure the functioningof) an expression control sequence. These expression vectors aretypically replicable in the host organisms either as episomes or as anintegral part of the host chromosomal DNA. Commonly, expression vectorswill contain selection markers, e.g., tetracycline or neomycin, topermit detection of those cells transformed with the desired DNAsequences (see, e.g., U.S. Pat. No. 4,704,362, which is incorporatedherein by reference).

Escherichia coli is one prokaryotic host useful for cloning thepolynucleotides of the present invention. Other microbial hosts suitablefor use include bacilli, such as Bacillus subtilis, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species.

Other eukaryotic cells may be used, including yeast cells, insect tissueculture cells, avian cells or the like. Preferably, mammalian tissuecell culture will be used to produce the polypeptides of the presentinvention (see, Winnacker, From Genes to Clones, VCH Publishers, N.Y.(1987), which is incorporated herein by reference). Yeast and mammaliancells are preferred cells in which to use leafhopper ecdysonereceptor-based inducible gene expression systems because they naturallylack the molecules which confer responsiveness to the ligands forecdysone receptor.

Expression vectors may also include expression control sequences, suchas an origin of replication, a promoter, an enhancer, a responseelement, and necessary processing information sites, such asribosome-binding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferably, the enhancers orpromoters will be those naturally associated with genes encoding thesteroid receptors, although it will be understood that in many casesothers will be equally or more appropriate. Other preferred expressioncontrol sequences are enhancers or promoters derived from viruses, suchas SV40, Adenovirus, Bovine Papilloma Virus, and the like.

The vectors comprising the polynucleotides of the present invention canbe transferred into the host cell by well-known methods, which varydepending on the type of cellular host. For example, calcium chloridetransfection is commonly utilized for prokaryotic cells, whereas calciumphosphate treatment may be used for other cellular hosts. (See,generally, Sambrook et al. (1989), Molecular Cloning: A LaboratoryManual (2d ed.), Cold Spring Harbor Press, which is incorporated hereinby reference). The term “transformed cell” is meant to also include theprogeny of a transformed cell.

The necessary transcriptional and translational signals can be providedon a recombinant expression vector, or they may be supplied by thenative gene encoding leafhopper ecdysone receptor and/or its flankingregions. Potential host-vector systems include but are not limited tomammalian cell systems infected with virus (e.g., vaccinia virus,adenovirus, etc.); insect cell systems infected with virus (e.g.,baculovirus); microorganisms such as yeast containing yeast vectors; orbacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmidDNA.

The expression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

A recombinant leafhopper ecdysone receptor protein of the invention, orfunctional fragment, derivative, chimeric construct, or analog thereof,may be expressed chromosomally, after integration of the coding sequenceby recombination. In this regard, any of a number of amplificationsystems may be used to achieve high levels of stable gene expression(See Sambrook et al., 1989, supra).

The cell into which the recombinant vector comprising the polynucleotideencoding leafhopper ecdysone receptor is cultured in an appropriate cellculture medium under conditions that provide for expression ofleafhopper ecdysone receptor by the cell. Any of the methods previouslydescribed for the insertion of DNA fragments into a cloning vector maybe used to construct expression vectors containing a gene consisting ofappropriate transcriptional/translational control signals and theprotein coding sequences. These methods may include in vitro recombinantDNA and synthetic techniques and in vivo recombination (geneticrecombination).

A polynucleotide encoding a leafhopper ecdysone receptor polypeptide maybe operably linked and controlled by any regulatory region, i.e.,promoter/enhancer element known in the art, but these regulatoryelements must be functional in the host cell selected for expression.The regulatory regions may comprise a promoter region for functionaltranscription in the host cell, as well as a region situated 3′ of thegene of interest, and which specifies a signal for termination oftranscription and a polyadenylation site. All these elements constitutean expression cassette.

Expression vectors comprising a polynucleotide encoding a leafhopperecdysone receptor polypeptide of the invention can be identified by fivegeneral approaches: (a) PCR amplification of the desired plasmid DNA orspecific mRNA, (b) nucleic acid hybridization, (c) presence or absenceof selection marker gene functions, (d) analyses with appropriaterestriction endonucleases, and (e) expression of inserted sequences. Inthe first approach, the nucleic acids can be amplified by PCR to providefor detection of the amplified product. In the second approach, thepresence of a foreign gene inserted in an expression vector can bedetected by nucleic acid hybridization using probes comprising sequencesthat are homologous to an inserted marker gene. In the third approach,the recombinant vector/host system can be identified and selected basedupon the presence or absence of certain “selection marker” genefunctions (e.g., β-galactosidase activity, thymidine kinase activity,resistance to antibiotics, transformation phenotype, occlusion bodyformation in baculovirus, etc.) caused by the insertion of foreign genesin the vector. In another example, if the nucleic acid encoding aleafhopper ecdysone receptor polypeptide is inserted within the“selection marker” gene sequence of the vector, recombinants comprisingthe leafhopper ecdysone receptor nucleic acid insert can be identifiedby the absence of the gene function. In the fourth approach, recombinantexpression vectors are identified by digestion with appropriaterestriction enzymes. In the fifth approach, recombinant expressionvectors can be identified by assaying for the activity, biochemical, orimmunological characteristics of the gene product expressed by therecombinant, provided that the expressed protein assumes a functionallyactive conformation.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includebut are not limited to derivatives of SV40 and known bacterial plasmids,e.g., E. coli plasmids col E1, pCR1, pBR322, pMal-C2, pET, pGEX (Smithet al., 1988, Gene 67: 31-40), pMB9 and their derivatives, plasmids suchas RP4; phage DNAS, e.g., the numerous derivatives of phage 1, e.g.,NM989, and other phage DNA, e.g., M13 and filamentous single strandedphage DNA; yeast plasmids such as the 2 m plasmid or derivativesthereof, vectors useful in eukaryotic cells, such as vectors useful ininsect or mammalian cells; vectors derived from combinations of plasmidsand phage DNAs, such as plasmids that have been modified to employ phageDNA or other expression control sequences; and the like.

The present invention also provides a gene expression cassette that iscapable of being expressed in a host cell, wherein the gene expressioncassette comprises a polynucleotide that encodes a leafhopper ecdysonereceptor polypeptide according to the invention. Thus, Applicants'invention also provides novel gene expression cassettes useful in anecdysone receptor-based gene expression system.

In a specific embodiment, the gene expression cassette that is capableof being expressed in a host cell comprises a polynucleotide thatencodes a polypeptide selected from the group consisting of a) apolypeptide comprising a transactivation domain, a DNA-binding domain,and a leafhopper ecdysone receptor ligand binding domain; b) apolypeptide comprising a DNA-binding domain and a leafhopper ecdysonereceptor ligand binding domain; and c) a polypeptide comprising atransactivation domain and a leafhopper ecdysone receptor ligand bindingdomain.

In another specific embodiment, the present invention provides a geneexpression cassette that is capable of being expressed in a host cell,wherein the gene expression cassette comprises a polynucleotide thatencodes a hybrid polypeptide selected from the group consisting of a) ahybrid polypeptide comprising a transactivation domain, a DNA-bindingdomain, and a leafhopper ecdysone receptor ligand binding domain; b) ahybrid polypeptide comprising a DNA-binding domain and a leafhopperecdysone receptor ligand binding domain; and c) a hybrid polypeptidecomprising a transactivation domain and a leafhopper ecdysone receptorligand binding domain. A hybrid polypeptide according to the inventioncomprises at least two polypeptide fragments, wherein each polypeptidefragment is from a different source, i.e., a different polypeptide, adifferent nuclear receptor, a different species, etc. The hybridpolypeptide according to the invention may comprise at least twopolypeptide domains, wherein each polypeptide domain is from a differentsource.

Preferably, the leafhopper ecdysone receptor ligand binding domain isfrom a green leafhopper Nephotetix cincticeps EcR (“NcEcR”).

In a specific embodiment, the leafhopper ecdysone receptor ligandbinding domain is encoded by a polynucleotide comprising a nucleic acidsequence selected from the group consisting of SEQ ID NO: 1, nucleotides16-1109 of SEQ ID NO: 1, nucleotides 214-1109 of SEQ ID NO: 1,nucleotides 448-1109 of SEQ ID NO: 1, and nucleotides 418-1109 of SEQ IDNO: 1.

In a specific embodiment, the leafhopper ecdysone receptor ligandbinding domain comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, amino acids 63-70 of SEQ ID NO: 2, aminoacids 72-370 of SEQ ID NO: 2, amino acids 150-370 of SEQ ID NO: 2, andamino acids 140-370 of SEQ ID NO: 2.

The DNA binding domain can be any DNA binding domain with a knownresponse element, including synthetic and chimeric DNA binding domains,or analogs, combinations, or modifications thereof. Preferably, the DBDis a GAL4 DBD, a LexA DBD, a transcription factor DBD, a steroid/thyroidhormone nuclear receptor superfamily member DBD, such as an EcR DBD, ora bacterial LacZ DBD.

The transactivation domain (abbreviated “AD” or “TA”) may be anysteroid/thyroid hormone nuclear receptor AD, synthetic or chimeric AD,polyglutamine AD, basic or acidic amino acid AD, a VP16 AD, a GAL4 AD,an NF-κB AD, a BP64 AD, a B42 acidic activation domain (B42AD), a p65transactivation domain (p65AD), or an analog, combination, ormodification thereof. In a specific embodiment, the AD is a synthetic orchimeric AD, or is obtained from an EcR, a glucocorticoid receptor,VP16, GAL4, NF-κB, or B42 acidic activation domain AD.

In a specific embodiment, the gene expression cassette encodes a hybridpolypeptide comprising either a) a DNA-binding domain, or b) atransactivation domain; and an NcEcR ligand binding domain according tothe invention.

The present invention also provides a gene expression cassettecomprising: i) a response element comprising a domain recognized by apolypeptide comprising a DNA binding domain; ii) a promoter that isactivated by a polypeptide comprising a transactivation domain; and iii)a gene whose expression is to be modulated.

The response element (“RE”) may be any response element with a known DNAbinding domain, or an analog, combination, or modification thereof. Asingle RE may be employed or multiple REs, either multiple copies of thesame RE or two or more different REs, may be used in the presentinvention. In a specific embodiment, the RE is an RE from GAL4(“GAL4RE”), LexA, a steroid/thyroid hormone nuclear receptor RE, such asan ecdysone response element (EcRE), or a synthetic RE that recognizes asynthetic DNA binding domain.

A steroid/thyroid hormone nuclear receptor DNA binding domain,activation domain or response element according to the invention may beobtained from a steroid/thyroid hormone nuclear receptor selected fromthe group consisting of ecdysone receptor (EcR), ubiquitous receptor(UR), Orphan receptor 1 (OR-1), steroid hormone nuclear receptor 1(NER-1), RXR interacting protein-15 (RIP-15), liver x receptor β (LXRβ),steroid hormone receptor like protein (RLD-1), liver x receptor (LXR),liver x receptor α (LXRα), farnesoid x receptor (FXR), receptorinteracting protein 14 (RIP-14), farnesol receptor (HRR-1), thyroidhormone receptor α (TRα), thyroid receptor 1 (c-erbA-1), thyroid hormonereceptor β (TRβ), retinoic acid receptor a (RARα), retinoic acidreceptor β (RARβ, HAP), retinoic acid receptor γ (RARγ), retinoic acidreceptor gamma-like (RARD), peroxisome proliferator-activated receptor α(PPARα), peroxisome proliferator-activated receptor β (PPARβ),peroxisome proliferator-activated receptor δ (PPARδ, NUC-1), peroxisomeproliferator-activator related receptor (FFAR), peroxisomeproliferator-activated receptor γ (PPARγ), orphan receptor encoded bynon-encoding strand of thyroid hormone receptor α (REVERBα), v-erb Arelated receptor (EAR-1), v-erb related receptor (EAR-1A), γ), orphanreceptor encoded by non-encoding strand of thyroid hormone receptor β(REVERBβ), verb related receptor (EAR-1β), orphan nuclear receptor BD73(BD73), rev-erbA-related receptor (RVR), zinc finger protein 126 (HZF2),ecdysone-inducible protein E75 (E75), ecdysone-inducible protein E78(E78), Drosophila receptor 78 (DR-78), retinoid-related orphan receptorα (RORα), retinoid Z receptor α (RZRα), retinoid related orphan receptorβ (RORβ), retinoid Z receptor β (RZRβ), retinoid-related orphan receptorγ (RORγ), retinoid Z receptor γ (RZRγ), retinoid-related orphan receptor(TOR), hormone receptor 3 (HR-3), Drosophila hormone receptor 3 (DHR-3),Manduca hormone receptor 3 (MHR-3), Gallaria hormone receptor 3 (GHR-3),C. elegans nuclear receptor 3 (CNR-3), Choristoneura hormone receptor 3(CHR-3), C. elegans nuclear receptor 14 (CNR-14), vitamin D receptor(VDR), orphan nuclear receptor (ONR-1), pregnane X receptor (PXR),steroid and xenobiotic receptor (SXR), benzoate X receptor (BXR),nuclear receptor (MB-67), constitutive androstane receptor 1 (CAR-1),constitutive androstane receptor α (CARα), constitutive androstanereceptor 2 (CAR-2), constitutive androstane receptor β (CARβ),Drosophila hormone receptor 96 (DHR-96), nuclear hormone receptor 1(NHR-1), hepatocyte nuclear factor 4 (HNF-4), hepatocyte nuclear factor4G (HNF-4G), hepatocyte nuclear factor 4B (HNF-4B), hepatocyte nuclearfactor 4D (HNF-4D, DHNF-4), retinoid X receptor α (RXRα), retinoid Xreceptor β (RXRβ), H-2 region II binding protein (H-2RIIBP), nuclearreceptor co-regulator-1 (RCoR-1), retinoid X receptor γ (RXRγ),Ultraspiracle (USP), 2C1 nuclear receptor, chorion factor 1 (CF-1),testicular receptor 2 (TR-2), testicular receptor 2-11 (TR2-11),testicular receptor 4 (TR4), TAK-1, Drosophila hormone receptor (DHR78),Tailless (ILL), tailless homolog (TLX), XTLL, chicken ovalbumin upstreampromoter transcription factor I (COUP-TFI), chicken ovalbumin upstreampromoter transcription factor A (COUP-TFA), EAR-3, SVP-44, chickenovalbumin upstream promoter transcription factor II (COUP-TFII), chickenovalbumin upstream promoter transcription factor B (COUP-TFB), ARP-1,SVP40, SVP, chicken ovalbumin upstream promoter transcription factor III(COUP-TFIII), chicken ovalbumin upstream promoter transcription factor G(COUP-TFG), SVP-46, EAR-2, estrogen receptor α (ERα), estrogen receptorβ (FRβ), estrogen related receptor 1 (ERR1), estrogen related receptor α(ERRα), estrogen related receptor 2 (ERR2), estrogen related receptor β(ERRβ), glucocorticoid receptor (GR), mineralocorticoid receptor (MR),progesterone receptor (PR), androgen receptor (AR), nerve growth factorinduced gene B (NGFI-B), nuclear receptor similar to Nur-77 (TRS), N10,Orphan receptor (NUR-77), Human early response gene (NAK-1), Nurrrelated factor 1 (NURR-1), a human immediate-early response gene (NOT),regenerating liver nuclear receptor 1 (RNR-1), hematopoietic zinc finger3 (HZF-3), Nur related protein-1 (TINOR), Nuclear orphan receptor 1(NOR-1), NOR1 related receptor (MINOR), Drosophila hormone receptor 38(DHR-38), C. elegans nuclear receptor 8 (CNR-8), C48D5, steroidogenicfactor 1 (SF1), endozepine-like peptide (ELP), fushi tarazu factor 1(FTZ-F1), adrenal 4 binding protein (AD4BP), liver receptor homolog(LRH-1), Ftz-F1-related orphan receptor A (xFFrA), Ftz-F1-related orphanreceptor B (xFFrB), nuclear receptor related to LRH-1 (FFLR), nuclearreceptor related to LRH-1 (PHR), fetoprotein transcription factor (FTF),germ cell nuclear factor (GCNFM), retinoid receptor-relatedtestis-associated receptor (RTR), knirps (KNI), knirps related (KNRL),Embryonic gonad (EGON), Drosophila gene for ligand dependent nuclearreceptor (EAGLE), nuclear receptor similar to trithorax (ODR7),Trithorax, dosage sensitive sex reversal adrenal hypoplasia congenitacritical region chromosome X gene (DAX-1), adrenal hypoplasia congenitaand hypogonadotropic hypogonadism (AHCH), and short heterodimer partner(SHP).

For purposes of this invention, nuclear receptors and leafhopperecdysone receptors also include synthetic and chimeric nuclear receptorsand leafhopper ecdysone receptors and their homologs.

Antibodies to Leafhopper Ecdysone Receptor

According to the invention, a leafhopper ecdysone receptor polypeptideproduced recombinantly or by chemical synthesis, and fragments or otherderivatives or analogs thereof, including fusion proteins, may be usedas an antigen or immunogen to generate antibodies. Preferably, theantibodies specifically bind homopteran ecdysone receptor polypeptides,but do not bind other ecdysone receptor polypeptides. More preferably,the antibodies specifically bind a leafhopper ecdysone receptorpolypeptide, but do not bind other ecdysone receptor polypeptides.

The present invention also relates to antigenic peptides and antibodiesthereto. More particularly, the invention relates to antigenic peptidescomprising a fragment of a leafhopper ecdysone receptor polypeptideaccording to the invention, wherein the fragment has a property selectedfrom the group consisting of:

(a) it is encoded by a polynucleotide comprising a nucleic acid sequenceselected from the group consisting of nucleotides 1-15 of SEQ ID NO: 1,nucleotides 16-213 of SEQ ID NO: 1, nucleotides 214-447 of SEQ ID NO: 1,nucleotides 448-1109 of SEQ ID NO: 1, nucleotides 16-1109 of SEQ ID NO:1, nucleotides 214-1109 of SEQ ID NO: 1, and nucleotides 418-1109 of SEQID NO: 1;

(b) it comprises an amino acid sequence selected from the groupconsisting of amino acids 1-5 of SEQ ID NO: 2, amino acids 6-71 of SEQID NO: 2, amino acids 72-149 of SEQ ID NO: 2, amino acids 150-370 of SEQID NO: 2, amino acids 6-370 of SEQ ID NO: 2, amino acids 72-370 of SEQID NO: 2, and amino acids 140-370 of SEQ ID NO: 2; and

(c) it specifically binds to an antibody generated against an epitopewithin a polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2, amino acids 1-5 of SEQ ID NO: 2, aminoacids 6-71 of SEQ ID NO: 2, amino acids 72-149 of SEQ ID NO: 2, aminoacids 150-370 of SEQ ID NO: 2, amino acids 6-370 of SEQ ID NO: 2, aminoacids 72-370 of SEQ ID NO: 2, and amino acids 140-370 of SEQ ID NO: 2.

In another embodiment, the invention relates to an antibody whichspecifically binds an antigenic peptide comprising a fragment of aleafhopper ecdysone receptor polypeptide according to the invention asdescribed above. The antibody may be polyclonal or monoclonal and may beproduced by in vitro or in vivo techniques.

The antibodies of the invention possess specificity for binding toparticular homopteran ecdysone receptors. Thus, reagents for determiningqualitative or quantitative presence of these or homologous polypeptidesmay be produced. Alternatively, these antibodies may be used to separateor purify receptor polypeptides.

For production of polyclonal antibodies, an appropriate target immunesystem is selected, typically a mouse or rabbit. The substantiallypurified antigen is presented to the immune system in a fashiondetermined by methods appropriate for the animal and other parameterswell known to immunologists. Typical sites for injection are in thefootpads, intramuscularly, intraperitoneally, or intradermally. Ofcourse, another species may be substituted for a mouse or rabbit.

An immunological response is usually assayed with an immunoassay.Normally such immunoassays involve some purification of a source ofantigen, for example, produced by the same cells and in the same fashionas the antigen was produced. The immunoassay may be a radioimmunoassay,an enzyme-linked assay (ELISA), a fluorescent assay, or any of manyother choices, most of which are functionally equivalent but may exhibitadvantages under specific conditions.

Monoclonal antibodies with high affinities are typically made bystandard procedures as described, e.g., in Harlow and Lane (1988),Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; orGoding (1986), Monoclonal Antibodies: Principles and Practice (2d ed)Academic Press, New York, which are hereby incorporated herein byreference. Briefly, appropriate animals will be selected and the desiredimmunization protocol followed. After the appropriate period of time,the spleens of such animals are excised and individual spleen cellsfused, typically, to immortalized myeloma cells under appropriateselection conditions. Thereafter, the cells are clonally separated andthe supernatants of each clone are tested for their production of anappropriate antibody specific for the desired region of the antigen.

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides or alternatively to selection of libraries ofantibodies in phage or similar vectors. See, Huse et al., (1989)“Generation of a Large Combinatorial Library of the ImmunoglobulinRepertoire in Phage Lambda,” Science 246: 1275-1281, hereby incorporatedherein by reference.

The polypeptides and antibodies of the present invention may be usedwith or without modification. Frequently, the polypeptides andantibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescence, chemiluminescence, magnetic particles and thelike. Patents, teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Also, recombinant immunoglobulins may be produced, seeCabilly, U.S. Pat. No.4,816,567.

A molecule is “antigenic” when it is capable of specifically interactingwith an antigen recognition molecule of the immune system, such as animmunoglobulin (antibody) or T cell antigen receptor. An antigenicpolypeptide contains at least about 5, and preferably at least about 10amino acids. An antigenic portion of a molecule can be that portion thatis immunodominant for antibody or T cell receptor recognition, or it canbe a portion used to generate an antibody to the molecule by conjugatingthe antigenic portion to a carrier molecule for immunization. A moleculethat is antigenic need not be itself immunogenic, i.e., capable ofeliciting an immune response without a carrier.

Such antibodies include but are not limited to polyclonal, monoclonal,chimeric, single chain, Fab fragments, and an Fab expression library.The anti-leafhopper ecdysone receptor antibodies of the invention may becross-reactive, e.g., they may recognize leafhopper ecdysone receptorfrom different species. Polyclonal antibodies have greater likelihood ofcross reactivity. Alternatively, an antibody of the invention may bespecific for a single form of leafhopper ecdysone receptor, such asleafhopper ecdysone receptor. Preferably, such an antibody is specificfor leafhopper ecdysone receptor.

Various procedures known in the art may be used for the production ofpolyclonal antibodies. For the production of antibody, various hostanimals can be immunized by injection with the leafhopper ecdysonereceptor polypeptide, or a derivative (e.g., fragment or fusion protein)thereof, including but not limited to rabbits, mice, rats, sheep, goats,etc. In one embodiment, the leafhopper ecdysone receptor polypeptide orfragment thereof can be conjugated to an immunogenic carrier, e.g.,bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Variousadjuvants may be used to increase the immunological response, dependingon the host species, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward the leafhopperecdysone receptor polypeptide, or fragment, analog, or derivativethereof, any technique that provides for the production of antibodymolecules by continuous cell lines in culture may be used. These includebut are not limited to the hybridoma technique originally developed byKohler and Milstein [Nature 256:495-497 (1975)], as well as the triomatechnique, the human B-cell hybridoma technique [Kozbor et al.,Immunology Today 4: 72 1983); Cote et al., Proc. Natl. Acad Sci. U.S.A.80: 2026-2030 (1983)], and the EBV-hybridoma technique to produce humanmonoclonal antibodies [Cole et al., in Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96 (1985)]. In an additionalembodiment of the invention, monoclonal antibodies can be produced ingerm-free animals [International Patent Publication No. WO 89/12690,published 28 Dec. 1989]. In fact, according to the invention, techniquesdeveloped for the production of “chimeric antibodies” [Morrison et al.,J. Bacteriol. 159:870 (1984); Neuberger et al., Nature 312: 604-608(1984); Takeda et al., Nature 314: 452-454 (1985)] by splicing the genesfrom a mouse antibody molecule specific for a leafhopper ecdysonereceptor polypeptide together with genes from a human antibody moleculeof appropriate biological activity can be used; such antibodies arewithin the scope of this invention Such human or humanized chimericantibodies are preferred for use in therapy of human diseases ordisorders (described infra), since the human or humanized antibodies aremuch less likely than xenogenic antibodies to induce an immune response,in particular an allergic response, themselves.

According to the invention, techniques described for the production ofsingle chain Fv (scFv) antibodies [U.S. Pat. Nos. 5,476,786 and5,132,405 to Huston; U.S. Pat. No. 4,946,778] can be adapted to produceleafhopper ecdysone receptor polypeptide-specific single chainantibodies. An additional embodiment of the invention utilizes thetechniques described for the construction of Fab expression libraries[Huse et al., Science 246: 1275-1281 (1989)] to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificityfor a leafhopper codysone receptor polypeptide, or its derivatives, oranalogs.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbent assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprint antibody is detected by detecting binding of a secondary antibodyor reagent to the primary antibody. In a further embodiment, thesecondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of a leafhopper ecdysone receptor polypeptide, one mayassay generated hybridomas for a product which binds to a leafhopperecdysone receptor polypeptide fragment containing such epitope. Forselection of an antibody specific to a leafhopper ecdysone receptorpolypeptide from a particular species of animal, one can select on thebasis of positive binding with leafhopper ecdysone receptor polypeptideexpressed by or isolated from cells of that species of animal.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the leafhopper ecdysonereceptor polypeptide, e.g., for western blotting, imaging leafhopperecdysone receptor polypeptide in situ, measuring levels thereof inappropriate physiological samples, etc. using any of the detectiontechniques mentioned above or known in the art.

In a specific embodiment, antibodies that agonize or antagonize theactivity of leafhopper ecdysone receptor polypeptide can be generated.Such antibodies can be tested using the assays described infra foridentifying ligands. In particular, such antibodies can be scFvantibodies expressed intracellularly.

Uses of Novel Polynucleotides and Polypeptides of the Invention

The present invention further provides a number of uses for theleafhopper ecdysone receptor polynucleotides of the present inventionand their encoded polypeptides.

The leafhopper ecdysone receptor polypeptides of the present inventionhave a variety of utilities. For example, the polynucleotides andpolypeptides of the invention are useful in methods of modulating geneexpression in an ecdysone receptor-based gene expression system. Alsoincluded are methods for identifying and selecting ligands specific forbinding to a ligand binding domain of a polypeptide of the invention,methods for identifying and selecting compounds exhibiting specificbinding to the ligand binding domain and methods for modulating insectphysiology or development (e.g., killing).

Methods of Modulating Gene Expression

As presented herein, Applicants' novel polynucleotides and polypeptidesare useful in an ecdysone receptor-based gene expression system toprovide a regulatable gene expression system in both prokaryotic andeukaryotic host cells. Thus, the present invention also relates to theuse of the novel leafhopper ecdysone receptor polynucleotides andpolypeptides of the present invention in an ecdysone receptor-based geneexpression system, and methods of modulating the expression of a genewithin a host cell using such an ecdysone receptor-based gene expressionsystem.

This gene expression system may be a “single switch”-based geneexpression system in which the transactivation domain, DNA-bindingdomain and ligand binding domain are on one encoded polypeptide.Alternatively, the gene expression modulation system may be a “dualswitch”- or “two-hybrid”-based gene expression modulation system inwhich the transactivation domain and DNA-binding domain are located ontwo different encoded polypeptides. Applicants′ have demonstrated forthe first time that leafhopper ecdysone receptor polynucleotides andpolypeptides of the invention can be used as a component of an ecdysonereceptor-based inducible gene expression system to modify geneexpression in a host cell.

In particular, the present invention relates to a gene expressionmodulation system comprising at least one gene expression cassette thatis capable of being expressed in a host cell comprising a polynucleotidethat encodes a leafhopper ecdysone receptor polypeptide. Preferably, theleafhopper ecdysone receptor polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 2, amino acids1-5 of SEQ ID NO: 2, amino acids 6-71 of SEQ ID NO: 2, amino acids72-149 of SEQ ID NO: 2, amino acids 150-370 of SEQ ID NO: 2, amino acids6-370 of SEQ ID NO: 2, amino acids 72-370 of SEQ ID NO: 2, and aminoacids 140-370 of SEQ ID NO: 2. More preferably, the leafhopper ecdysonereceptor polypeptide comprises amino acids 6-370 of SEQ ID NO: 2, aminoacids 72-370 of SEQ ID NO: 2, amino acids 150-370 of SEQ ID NO: 2, oramino acids 140-370 of SEQ ID NO: 2.

In a specific embodiment, the gene expression modulation systemcomprises a gene expression cassette comprising a polynucleotide thatencodes a polypeptide comprising a transactivation domain, a DNA-bindingdomain that recognizes a response element associated with a gene whoseexpression is to be modulated; and a leafhopper ecdysone receptor ligandbinding domain (referred to herein as “NcEcR LBD”). The gene expressionmodulation system may further comprise a second gene expression cassettecomprising: i) a response element recognized by the DNA-binding domainof the encoded polypeptide of the first gene expression cassette; ii) apromoter that is activated by the transactivation domain of the encodedpolypeptide of the first gene expression cassette; and iii) a gene whoseexpression is to be modulated.

In another specific embodiment, the gene expression modulation systemcomprises a gene expression cassette comprising a) a polynucleotide thatencodes a polypeptide comprising a transactivation domain, a DNA-bindingdomain that recognizes a response element associated with a gene whoseexpression is to be modulated; and an NcEcR LBD, and b) a second nuclearreceptor ligand binding domain selected from the group consisting of avertebrate retinoid X receptor ligand binding domain, an invertebrateretinoid X receptor ligand binding domain, an ultraspiracle proteinligand binding domain, and a chimeric ligand binding domain comprisingtwo polypeptide fragments, wherein the first polypeptide fragment isfrom a vertebrate retinoid X receptor ligand binding domain, aninvertebrate retinoid X receptor ligand binding domain, or anultraspiracle protein ligand binding domain, and the second polypeptidefragment is from a different vertebrate retinoid X receptor ligandbinding domain, invertebrate retinoid X receptor ligand binding domain,or ultraspiracle protein ligand binding domain. The gene expressionmodulation system may further comprise a second gene expression cassettecomprising: i) a response element recognized by the DNA-binding domainof the encoded polypeptide of the first gene expression cassette; ii) apromoter that is activated by the transactivation domain of the encodedpolypeptide of the first gene expression cassette; and iii) a gene whoseexpression is to be modulated.

In another specific embodiment, the gene expression modulation systemcomprises a first gene expression cassette comprising a polynucleotidethat encodes a first polypeptide comprising a DNA-binding domain thatrecognizes a response element associated with a gene whose expression isto be modulated and a nuclear receptor ligand binding domain, and asecond gene expression cassette comprising a polynucleotide that encodesa second polypeptide comprising a transactivation domain and a nuclearreceptor ligand binding domain, wherein one of the nuclear receptorligand binding domains is an NcEcR LBD. In a preferred embodiment, thefirst polypeptide is substantially free of a transactivation domain andthe second polypeptide is substantially free of a DNA binding domain.For purposes of the invention, “substantially free of a DNA bindingdomain” means that the protein in question does not contain a sufficientsequence of the domain in question to provide activation or bindingactivity. The gene expression modulation system may further comprise athird gene expression cassette comprising: i) a response elementrecognized by the DNA-binding domain of the first polypeptide of thefirst gene expression cassette; ii) a promoter that is activated by thetransactivation domain of the second polypeptide of the second geneexpression cassette; and iii) a gene whose expression is to bemodulated.

Wherein when only one nuclear receptor ligand binding domain is an NcEcRLBD, the other nuclear receptor ligand binding domain may be from anyother nuclear receptor that forms a dimer with the NcEcR LBD. Forexample, the other nuclear receptor ligand binding domain (“partner”)may be from another ecdysone receptor, a vertebrate retinoid X receptor(RXR), an invertebrate RXR, an ultraspiracle protein (USP), or achimeric nuclear receptor comprising at least two different nuclearreceptor ligand binding domain polypeptide fragments selected from thegroup consisting of a vertebrate RXR, an invertebrate RXR, and a USP(see co-pending applications PCT/US01/09050, U.S. 60/294,814, and U.S.60/294,819, incorporated herein by reference in their entirety). The“partner” nuclear receptor ligand binding domain may further comprise atruncation mutation, a deletion mutation, a substitution mutation, oranother modification.

In a specific embodiment, the leafhopper ecdysone receptor ligandbinding domain comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, amino acids 150-370 of SEQ ID NO: 2, aminoacids 6-370 of SEQ ID NO: 2, amino acids 72-370 of SEQ ID NO: 2, andamino acids 140-370 of SEQ ID NO: 2. In another embodiment, theleafhopper ecdysone receptor ligand binding domain is encoded by apolynucleotide comprises a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1, nucleotides 448-1109 of SEQ ID NO: 1,nucleotides 16-1109 of SEQ ID NO: 1, nucleotides 214-1109 of SEQ ID NO:1, and nucleotides 418-1109 of SEQ ID NO: 1.

In a specific embodiment, the gene whose expression is to be modulatedis a homologous gene with respect to the host cell. In another specificembodiment, the gene whose expression is to be modulated is aheterologous gene with respect to the host cell.

The ligands for use in the methods of modulating gene expression aredescribed below, when combined with the ligand binding domain of thenuclear receptor(s), which in turn are bound to the response elementlinked to a gene, provide the means for external temporal regulation ofexpression of the gene. The binding mechanism or the order in which thevarious components of this invention bind to each other, that is, forexample, ligand to ligand binding domain, DNA-binding domain to responseelement, transactivation domain to promoter, etc., is not critical.

Thus, Applicants' invention is useful in methods of modulating geneexpression in a host cell using a leafhopper ecdysone receptor accordingto the invention. Specifically, Applicants' invention provides a methodof modulating the expression of a gene in a host cell comprising thesteps of: a) introducing into the host cell an ecdysone receptor-basedgene expression modulation system comprising a leafhopper ecdysonereceptor according to the invention; and b) introducing into the hostcell a ligand; wherein the gene to be modulated is a component of a geneexpression cassette comprising: i) a response element comprising adomain recognized by the DNA binding domain of the gene expressionsystem; ii) a promoter that is activated by the transactivation domainof the gene expression system; and iii) a gene whose expression is to bemodulated, whereby upon introduction of the ligand into the host cell,expression of the gene is modulated.

The invention also provides a method of modulating the expression of agene in a host cell comprising the steps of: a) introducing into thehost cell an ecdysone receptor-based gene expression modulation systemcomprising a leafhopper ecdysone receptor according to the invention; b)introducing into the host cell a gene expression cassette, wherein thegene expression cassette comprises i) a response element comprising adomain recognized by the DNA binding domain from the gene expressionsystem; ii) a promoter that is activated by the transactivation domainof the gene expression system; and iii) a gene whose expression is to bemodulated; and c) introducing into the host cell a ligand; whereby uponintroduction of the ligand into the host cell, expression of the gene ismodulated.

Genes of interest for expression in a host cell using Applicants'methods may be endogenous genes or heterologous genes. Nucleic acid oramino acid sequence information for a desired gene or protein can belocated in one of many public access databases, for example, GENBANK,EMBL, Swiss-Prot, and PM, or in many biology related journalpublications. Thus, those skilled in the art have access to nucleic acidsequence information for virtually all known genes. Such information canthen be used to construct the desired constructs for the insertion ofthe gene of interest within the gene expression cassettes used in themethods described herein.

Examples of genes of interest for expression in a host cell using thesemethods include, but are not limited to: antigens produced in plants asvaccines, enzymes like alpha-amylase, phytase, glucanes, xylase andxylanase, genes for resistance against insects, nematodes, fungi,bacteria, viruses, and abiotic stresses, nutraceuticals,pharmaceuticals, vitamins, genes for modifying amino acid content,herbicide resistance, cold, drought, and heat tolerance, industrialproducts, oils, protein, carbohydrates, antioxidants, male sterileplants, flowers, fuels, other output traits, genes encodingtherapeutically desirable polypeptides or products that may be used totreat a condition, a disease, a disorder, a dysfunction, a geneticdefect, such as monoclonal antibodies, enzymes, proteases, cytokines,interferons, insulin, erthropoietin, clotting factors, other bloodfactors or components, viral vectors for gene therapy, virus forvaccines, targets for drug discovery, functional genomics, andproteomics analyses and applications, and the like.

The term “ligand” is meant herein to refer to a molecule that binds thedomain described here as the “ligand binding domain” Also, a ligand fora leafhopper ecdysone receptor is a ligand which serves either as thenatural ligand to which the ecdysone receptor binds, or a functionalanalogue which may serve as an agonist or antagonist.

Acceptable ligands are any that modulate expression of the gene whenbinding of the DNA binding domain of the gene expression systemaccording to the invention to the response element in the presence ofthe ligand results in activation or suppression of expression of thegenes. Preferred ligands include an ecdysteroid, such as ecdysone,20-hydroxyecdysone, ponasterone A, muristerone A, and the like,9-cis-retinoic acid, synthetic analogs of retinoic acid,N,N′-diacylhydrazines such as those disclosed in U.S. Pat. No.6,013,836; 5,117,057; 5,530,028; and 5,378,726; dibenzoylalkylcyanohydrazines such as those disclosed in European Application No.461,809; N-alkyl-N,N′-diaroylhydrazines such as those disclosed in U.S.Pat. No.5,225,443; N-acyl-N-alkylcarbonylhydrazines such as thosedisclosed in European Application No. 234,994;N-aroyl-N-alkyl-N′-aroylhydrazines such as those described in U.S. Pat.No. 4,985,461; each of which is incorporated herein by reference andother similar materials including3,5-di-tert-butylhydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide, andthe like.

In a preferred embodiment, the ligand for use in the method ofmodulating expression of gene is a compound of the formula:

wherein:

-   -   E is a (C₄-C₆)alkyl containing a tertiary carbon or a        cyano(C₃-C₅)alkyl containing a tertiary carbon;    -   R¹ is H, Me, Et, i-Pr, F, formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl,        CH₂OH, CH₂OMe, CH₂CN, CN, C°CH, 1-propynyl, 2-propynyl, vinyl,        OH, OMe, OEt, cyclopropyl, CF₂CF₃, CH═CHCN, allyl, azido, SCN,        or SCHF₂;    -   R² is H, Me, Et, n-Pr, i-Pr, formyl, CF₃, CHF₂, CHCl₂, CH₂F,        CH₂Cl, CH₂OH, CH₂OMe, CH₂CN, CN, C°CH, 1-propynyl, 2-propynyl,        vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe₂, NEt₂, SMe,        SEt, SOCF₃, OCF₂CF₂H, COEt, cyclopropyl, CF₂CF₃, CH═CHCN, allyl,        azido, OCF₃, OCHF₂, O-i-Pr, SCN, SCHF₂, SOMe, NH—CN, or joined        with R³ and the phenyl carbons to which R² and R³ are attached        to form an ethylenedioxy, a dihydrofuryl ring with the oxygen        adjacent to a phenyl carbon, or a dihydropyryl ring with the        oxygen adjacent to a phenyl carbon;    -   R³ is H, Et, or joined with R² and the phenyl carbons to which        R² and R³ are attached to form an ethylenedioxy, a dihydrofuryl        ring with the oxygen adjacent to a phenyl carbon, or a        dihydropyryl ring with the oxygen adjacent to a phenyl carbon;    -   R⁴, R⁵, and R⁶ are independently H, Me, Et, F, Cl, Br, formyl,        CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CN, C°CH, 1-propynyl,        2-propynyl, vinyl, OMe, OEt, SMe, or SEt.

In another preferred embodiment, the ligand for use in the method ofmodulating expression of gene is an ecdysone, 20-hydroxyecdysone,ponasterone A, or muristerone A.

In another preferred embodiment, a second ligand may be used in additionto the first ligand discussed above in the method of modulatingexpression of a gene. Preferably, this second ligand is 9-cis-retinoicacid or a synthetic analog of retinoic acid.

Screening Assays

Identification and isolation of a polynucleotide encoding a leafhopperecdysone receptor polypeptide of the invention provides for expressionof leafhopper ecdysone receptor in quantities greater than can beisolated from natural sources, or in indicator cells that are speciallyengineered to indicate the activity of leafhopper ecdysone receptorexpressed after transfection or transformation of the cells.Accordingly, in addition to rational design of agonists and antagonistsbased on the structure of leafhopper ecdysone receptor polypeptide, thepresent invention contemplates an alternative method for identifyingspecific ligands of leafhopper ecdysone receptor using various screeningassays known in the art.

Thus, the present invention also relates to methods of screening for acompound that induces or represses transactivation of a leafhopperecdysone receptor polypeptide in a cell by contacting a leafhopperecdysone receptor polypeptide with a candidate molecule and detectingreporter gene activity in the presence of the ligand. Candidatecompounds may be either agonists or antagonists of the leafhopperecdysone receptor polypeptide. In a preferred embodiment, the leafhopperecdysone receptor polypeptide is expressed from a polynucleotide in thecell and the transactivation activity (i.e., expression or repression ofa reporter gene) or compound binding activity is measured.

In a specific embodiment, the present invention relates to methods ofscreening for molecules that stimulate or inhibit leafhopper ecdysonereceptor activity in a cell by contacting a leafhopper ecdysone receptorpolypeptide with a candidate molecule and detecting leafhopper ecdysonereceptor activity in the presence of the molecule. Candidate moleculesmay be either agonists or antagonists of leafhopper ecdysone receptor.In a preferred embodiment, the leafhopper ecdysone receptor is expressedfrom a polynucleotide in the cell and the leafhopper ecdysone receptoractivity measured is by induction of expression or transactivation of areporter gene. Induction of reporter gene expression can be measured asdescribed herein.

Thus, one aspect of the present invention is a method for selectingmolecules or ligands that modulate the activity of a leafhopper ecdysonereceptor polypeptide. In a specific embodiment, the present inventionprovides a method for identifying a ligand specific for binding to aligand binding domain of a leafhopper ecdysone receptor comprising

(a) combining (i) a hybrid polypeptide comprising a leafhopper ecdysonereceptor ligand binding domain and a DNA binding domain from a steroidhormone nuclear receptor superfamily; and (ii) a polynucleotide encodinga second polypeptide, wherein the polynucleotide is operably linked to atranscriptional control element that is responsive to the DNA bindingdomain of the hybrid polypeptide;

(b) exposing the hybrid polypeptide and the polynucleotide of (a) to acompound;

(c) determining ligand activity of the compound of (b) by determininginduction of expression of the second polypeptide; and

(d) identifying the compound that results in the induction of expressionof the second polypeptide.

The present invention is also useful to search for orthogonal ligandsand orthogonal receptor-based gene expression systems such as thosedescribed in co-pending U.S. application 60/237,446, which isincorporated herein by reference in its entirety.

The ligand binding domain (“LBD”) of the ecdysone receptor, specificallybinds steroid and non-steroidal agonist ligands, thereby providing ameans to screen for new molecules possessing the property of bindingwith high affinity to the ligand binding domain. Thus, the ligandbinding domain of a leafhopper ecdysone receptor polypeptide may be usedas a reagent to develop a binding assay. On one level, the LBD can beused as an affinity reagent for a batch or in a column selectiveprocess, to selectively retain ligands which bind. Alternatively, afunctional assay is preferred for its greater sensitivity to ligandbinding. By using a reporter molecule for binding, either through adirect assay for binding, or through an expression or other functionallinkage between binding and another function, an assay for binding maybe developed. For example, by operable linkage of an easily assayablereporter gene to a controlling element responsive to binding by anecdysone receptor, and where ligand binding is functionally linked toprotein induction, an extremely sensitive assay for the presence of aligand or of a receptor results. Such a construct is useful for assayingthe presence of 20-hydroxyecdysone is described below. This construct isuseful for screening for agonists or antagonists of homopteran ecdysonereceptors, in particular, leafhopper ecdysone receptors.

As presented herein, a leafhopper ecdysone receptor can transactivategene expression of an ecdysone receptor-based gene expression modulationsystem. Therefore, agonists of leafhopper ecdysone receptor that enhanceits ability to transactivate gene expression will be expected to improveits activity in an ecdysone receptor-based gene expression modulationsystem. Inhibitors (antagonists) of leafhopper ecdysone receptoractivity are useful to reduce its ability to transactivate an ecdysonereceptor-based gene expression modulation system.

Any screening technique known in the art can be used to screen forleafhopper ecdysone receptor agonists or antagonists. For example, asuitable cell line expressing both leafhopper ecdysone receptor and anecdysone receptor-based gene expression modulation system can betransfected with a nucleic acid encoding a marker gene, such asβ-galactosidase. Cells are then exposed to a test solution comprising anagonist or antagonist, and then stained for β-galactosidase activity.The presence of more β-gal positive cells relative to control cells notexposed to the test solution is an indication of the presence of aleafhopper ecdysone receptor agonist in the test solution. Conversely,the presence of less β-gal positive cells relative to control cells notexposed to the test solution is an indication of the presence of aleafhopper ecdysone receptor antagonist in the test solution.

The present invention contemplates screens for small molecule ligands orligand analogs and mimics, as well as screens for natural ligands thatbind to and agonize or antagonize leafhopper ecdysone receptor in vivo.For example, natural products libraries can be screened using assays ofthe invention for molecules that agonize or antagonize leafhopperecdysone receptor activity.

Knowledge of the primary sequence of leafhopper ecdysone receptor, andthe similarity of that sequence with proteins of known function, canprovide an initial clue as the inhibitors or antagonists of the protein.Identification and screening of antagonists is further facilitated bydetermining structural features of the protein, e.g., using X-raycrystallography, neutron diffraction, nuclear magnetic resonancespectrometry, and other techniques for structure determination. Thesetechniques provide for the rational design or identification of agonistsand antagonists.

Another approach uses recombinant bacteriophage to produce largelibraries. Using the “phage method” [Scott and Smith, 1990, Science 249:386-390 (1990); Cwirla, et al., Proc. Natl. Acad. Sci., 87: 6378-6382(1990); Devlin et al., Science, 249: 404-406 (1990)], very largelibraries can be constructed (10⁶-10⁸ chemical entities). A secondapproach uses primarily chemical methods, of which the Geysen method[Geysen et al., Molecular Immunology 23: 709-715 (1986); Geysen et al.J. Immunologic Method 102: 259-274 (1987)] and the method of Fodor etal. [Science 251: 767-773 (1991)] are examples, et al. [14thInternational Congress of Biochemistry, Volume 5, Abstract FR:013(1988); Furka, Int. J. Peptide Protein Res. 37: 487-493 (1991)],Houghton [U.S. Pat. No. 4,631,211, issued December 1986] and Rutter etal. [U.S. Pat. No. 5,010,175, issued Apr. 23, 1991] describe methods toproduce a mixture of peptides that can be tested as agonists orantagonists.

In another aspect, synthetic libraries [Needels et al., Proc. Natl.Acad. Sci. USA 90: 10700-4 (1993); Ohlmeyer et al., Proc. Natl. Acad.Sci. USA 90: 10922-10926 (1993); Laam et al., International PatentPublication No. WO 92/00252; Kocis et al., International PatentPublication No. WO 9428028, each of which is incorporated herein byreference in its entirety], and the like can be used to screen forleafhopper ecdysone receptor ligands according to the present invention.

The screening can be performed with recombinant cells that express theleafhopper ecdysone receptor, or alternatively, using purified protein,e.g., produced recombinantly, as described above. For example, labeled,soluble leafhopper ecdysone receptor can be used to screen libraries, asdescribed in the foregoing references.

In one embodiment, leafhopper ecdysone receptor may be directly labeled.In another embodiment, a labeled secondary reagent may be used to detectbinding of a leafhopper ecdysone receptor to a molecule of interest,e.g., a molecule attached to a solid phase support. Binding may bedetected by in situ formation of a chromophore by an enzyme label.Suitable enzymes include, but are not limited to, allaline phosphataseand horseradish peroxidase. In a further embodiment, a two-color assay,using two chromogenic substrates with two enzyme labels on differentacceptor molecules of interest, may be used. Cross-reactive and singlyreactive ligands may be identified with a two-color assay.

Other labels for use in the invention include colored latex beads,magnetic beads, fluorescent labels (e.g., fluorescene isothiocyanate(FITC), phycoerythrin (PE), Texas red (TR), rhodamine, free or chelatedlanthanide series salts, especially Eu³⁺, to name a few fluorophores),chemiluminescent molecules, radio-isotopes, or magnetic resonanceimaging labels. Two-color assays may be performed with two or morecolored latex beads, or fluorophores that emit at different wavelengths.Labeled may be detected visually or by mechanical/optical means.Mechanical/optical means include fluorescence activated sorting, i.e.,analogous to FACS, and micromanipulator removal means.

As exemplified herein, the level of a leafhopper ecdysone receptorpolypeptide can be evaluated by metabolic labeling of the proteins. Asthe metabolic labeling occurs during in vitro incubation of the tissuebiopsy in the presence of culture medium supplemented with[³⁵S]-methionine, the level of each of the markers detected may beaffected by the in vitro conditions. In addition to metabolic (orbiosynthetic) labeling with [³⁵S]-methionine, the invention furthercontemplates labeling with [¹⁴C]-amino acids and [³H]-amino acids (withthe tritium substituted at non-labile positions). Thus, a sample orlibrary of compounds can be directly analyzed after labeling of theproteins therein, e.g., by calorimetric staining using silver, gold,coomassie blue, or amido-schwartz, to mention a few techniques; isotopiclabeling, e.g., with [³²P]-orthophosphate, [¹²⁵I], [¹³¹I]; fluorescentor chemiluminescent tags; and immunological detection with labeledantibody or specific binding partner of a marker.

Modulating Insect Physiology or Development

The isolation of a leafhopper ecdysone receptor provides for isolationor screening of new ligands for receptor binding. Some of these willinterfere with, or disrupt, normal insect development. It may sometimesbe important to either accelerate or decelerate insect development, forinstance, in preparing sterile adults for release. Alternatively, incertain circumstances, a delay or change in the timing of developmentmay be lethal or may dramatically modify the ability of an insect toaffect an agricultural crop. Thus, naturally occurring, biodegradableand highly active molecules to disrupt the timing of insect developmentwill result.

The present invention provides a means for disrupting insect developmentwhere new ligand agonists or antagonists are discovered. These compoundsare prime candidates as agonists or antagonists to interfere with thenormal insect development. By application of new analogues of ligandsfor a leafhopper ecdysone receptor, it is possible to modify the normaltemporal sequence of developmental events. For example, acceleratinginsect development will minimize generation time. This may be veryimportant in circumstances where large numbers of insects are desiredfinally, for instance, in producing sterile males. Alternatively, it maybe useful to slow development in a pest infestation, such that theinsects reach destructive stages of development only after commercialcrops may have passed sensitive stages. In another commercialapplication, ligands discovered by methods provided by the presentinvention may be used to artificially maintain insects in a specificdevelopmental stage. The development of larvae may also be acceleratedto reach a particular developmental stage in their life cycle earlierthan naturally.

Other analogues of ligands for a leafhopper ecdysone receptor may beselected which, upon application, may be completely disruptive of normaldevelopment, leading to a lethal result and pest control. Indeed, theremay be new ligands for a leafhopper ecdysone receptor which may bespecies specific or may exhibit a particularly useful spectrum ofeffectiveness. The greater specificity of the ligands will allowavoidance of use of non-specific pesticides possessing undesireddeleterious ecological side effects. Furthermore, compounds havingstructures closely analogous to natural compounds may be susceptible tonatural mechanisms of biological degradation.

Thus, the present invention also provides a method for identifying andselecting compounds exhibiting specific binding to the ligand bindingdomain to modulate insect physiology or development (e.g., killing)comprising the steps of screening compounds for binding to a homopteranecdysone receptor, selecting compounds exhibiting said binding andadministering the ligand to a homopteran insect. In a specificembodiment, a method for modulating insect physiology or developmentcomprises the steps of screening compounds for binding to a leafhopperecdysone receptor, selecting compounds exhibiting said binding andadministering the ligand to a leafhopper.

Polypeptide Production

A purified leafhopper ecdysone receptor polypeptide of the invention isalso useful in a method for determining the structural and biosyntheticaspects of the purified leafhopper ecdysone receptor polypeptide.Structural studies of interactions of the ligand-binding domains withselected ligands may be performed by various methods. The preferredmethod for structural determination is X-ray crystallography but mayinclude various other forms of spectroscopy or chromatography. See,e.g., Connolly, M. L., J. Appl. Crystall. 16: 548 (1983); and Connolly,M. L., Science 221: 709 (1983), which are hereby incorporated herein byreference. For example, the structure of the interaction between ligandand ligand-binding domain may be determined to high resolution.

Having provided for the substantially pure polypeptides, biologicallyactive fragments thereof and recombinant polynucleotides encoding them,the present invention also provides cells comprising each of them. Byappropriate introduction techniques well known in the field, cellscomprising them may be produced. See, e.g., Sambrook et al. (1989).

Host Cells and Non-Human Organisms

Another aspect of the present invention involves cells comprising anisolated polynucleotide encoding a leafhopper ecdysone receptorpolypeptide of the present invention. In a specific embodiment, theinvention relates to an isolated host cell comprising a vectorcomprising a polynucleotide encoding a leafhopper ecdysone receptorpolypeptide of the present invention. The present invention also relatesto an isolated host cell comprising an expression vector according tothe invention. In another specific embodiment, the invention relates toan isolated host cell comprising a gene expression cassette comprising apolynucleotide encoding a leafhopper ecdysone receptor polypeptide ofthe present invention. In another specific embodiment, the inventionrelates to an isolated host cell transfected with a gene expressionmodulation system comprising a polynucleotide encoding a leafhopperecdysone receptor polypeptide of the present invention. In anotherspecific embodiment, the invention also provides an isolated host cellcomprising an ecdysone receptor-based gene expression system comprisinga leafhopper ecdysone receptor polypeptide according to the invention.In another specific embodiment, the invention relates to an isolatedhost cell comprising a leafhopper ecdysone receptor polypeptide of thepresent invention. In still another embodiment, the invention relates toa method for producing a leafhopper ecdysone receptor polypeptide,wherein the method comprises culturing an isolated host cell comprisinga polynucleotide encoding a leafhopper ecdysone receptor polypeptide ofthe present invention in culture medium under conditions permittingexpression of the polynucleotide encoding the leafhopper ecdysonereceptor polypeptide, and isolating the leafhopper ecdysone receptorpolypeptide from the culture.

As described above, the polypeptides of the present invention and thepolynucleotides encoding them may be used to modulate gene expression ina host cell. Expression in transgenic host cells may be useful for theexpression of various genes of interest. Applicants′ invention providesfor modulation of gene expression in prokaryotic and eukaryotic hostcells. Expression in transgenic host cells is useful for the expressionof various polypeptides of interest including but not limited toantigens produced in plants as vaccines, enzymes like alpha-amylase,phytase, glucanase, and xylanase, genes for resistance against insects,nematodes, fungi, bacteria, viruses, and abiotic stresses, antigens,nutraceuticals, pharmaceuticals, vitamins, genes for modifying aminoacid content, herbicide resistance, cold, drought, and heat tolerance,industrial products, oils, protein, carbohydrates, antioxidants, malesterile plants, flowers, fuels, other output traits, therapeuticpolypeptides, pathway intermediates; for the modulation of pathwaysalready existing in the host for the synthesis of new productsheretofore not possible using the host; cell based assays; functionalgenomics assays, biotherapeutic protein production, proteomics assays,and the like. Additionally the gene products may be useful forconferring higher growth yields of the host or for enabling analternative growth mode to be utilized.

In a specific embodiment, the isolated host cell is a prokaryotic hostcell or a eukaryotic host cell. In another specific embodiment, theisolated host cell is an invertebrate host cell or a vertebrate hostcell. Preferably, the isolated host cell is selected from the groupconsisting of a bacterial cell, a fungal cell, a yeast cell, a nematodecell, an insect cell, a fish cell, a plant cell, an avian cell, ananimal cell, and a mammalian cell. More preferably, the isolated hostcell is a yeast cell, a nematode cell, an insect cell, a plant cell, azebrafish cell, a chicken cell, a hamster cell, a mouse cell, a ratcell, a rabbit cell, a cat cell, a dog cell, a bovine cell, a goat cell,a cow cell, a pig cell, a horse cell, a sheep cell, a simian cell, amonkey cell, a chimpanzee cell, or a human cell.

Examples of preferred host cells include, but are not limited to, fungalor yeast species such as Aspergillus, Trichoderma, Saccharomyces,Pichia, Candida, Hansenula, or bacterial species such as those in thegenera Synechocystis, Synechococcus, Salmonella, Bacillus,Acinetobacter, Rhodococcus, Streptomyces, Escherichia, Pseudomonas,Methylomonas, Methylobacter, Alcaligenes, Synechocystis, Anabaena,Thiobacillus, Methanobacterium and Klebsiella; plant species selectedfrom the group consisting of an apple, Arabidopsis, bajra, banana,barley, beans, beet, blackgram, chickpea, chili, cucumber, eggplant,favabean, maize, melon, millet, mungbean, oat, okra, Panicum, papaya,peanut, pea, pepper, pigeonpea, pineapple, Phaseolus, potato, pumpkin,rice, sorghum, soybean, squash, sugarcane, sugarbeet, sunflower, sweetpotato, tea, tomato, tobacco, watermelon, and wheat; animal; andmammalian host cells.

In a specific embodiment, the isolated host cell is a yeast cellselected from the group consisting of a Sacclaromyces, a Pichia, and aCandida host cell.

In another specific embodiment, the isolated host cell is a Caenorhabduselegans nematode cell. In another specific embodiment, the isolated hostcell is a plant cell selected from the group consisting of an apple,Arabidopsis, bajra, banana, barley, beans, beet, blackgram, chickpea,chili, cucumber, eggplant, favabean, maize, melon, millet, mungbean,oat, okra, Panicum, papaya, peanut, pea, pepper, pigeonpea, pineapple,Phaseolus, potato, pumpkin, rice, sorghum, soybean, squash, sugarcane,sugarbeet, sunflower, sweet potato, tea, tomato, tobacco, watermelon,and wheat cell.

In another specific embodiment, the isolated host cell is a zebrafishcell.

In another specific embodiment, the isolated host cell is a chickencell.

In another specific embodiment, the isolated host cell is a mammaliancell selected from the group consisting of a hamster cell, a mouse cell,a rat cell, a rabbit cell, a cat cell, a dog cell, a bovine cell, a goatcell, a cow cell, a pig cell, a horse cell, a sheep cell, a monkey cell,a chimpanzee cell, and a human cell.

Host cell transformation is well known in the art and may be achieved bya variety of methods including but not limited to electroporation, viralinfection, plasmid/vector transfection, non-viral vector mediatedtransfection, Agrobacterium-mediated transformation, particlebombardment, and the like. Expression of desired gene products involvesculturing the transformed host cells under suitable conditions andinducing expression of the transformed gene. Culture conditions and geneexpression protocols in prolaryotic and eukaryotic cells are well knownin the art (see General Methods section of Examples). Cells may beharvested and the gene products isolated according to protocols specificfor the gene product.

In addition, a host cell may be chosen that modulates the expression ofthe transfected polynucleotide, or modifies and processes thepolypeptide product in a specific fashion desired. Different host cellshave characteristic and specific mechanisms for the translational andpost-translational processing and modification [e.g., glycosylation,cleavage (e.g. of signal sequence)] of proteins. Appropriate cell linesor host systems can be chosen to ensure the desired modification andprocessing of the foreign protein expressed. For example, expression ina bacterial system can be used to produce a non-glycosylated coreprotein product. However, a polypeptide expressed in bacteria may not beproperly folded. Expression in yeast can produce a glycosylated product.Expression in eukaryotic cells can increase the likelihood of “native”glycosylation and folding of a heterologous protein. Moreover,expression in mammalian cells can provide a tool for reconstituting, orconstituting, the polypeptide's activity. Furthermore, differentvector/host expression systems may affect processing reactions, such asproteolytic cleavages, to a different extent.

Applicants' invention also relates to a non-human organism comprising anisolated host cell according to the invention. In a specific embodiment,the non-human organism is a prokaryotic organism or a eukaryoticorganism. In another specific embodiment, the non-human organism is aninvertebrate organism or a vertebrate organism.

Preferably, the non-human organism is selected from the group consistingof a bacterium, a fungus, a yeast, a nematode, an insect, a fish, aplant, a bird, an animal, and a mammal. More preferably, the non-humanorganism is a yeast, a nematode, an insect, a plant, a zebrafish, achicken, a hamster, a mouse, a rat, a rabbit, a cat, a dog, a bovine, agoat, a cow, a pig, a horse, a sheep, a simian, a monkey, or achimpanzee.

In a specific embodiment, the non-human organism is a yeast selectedfrom the group consisting of Saccharomyces, Pichia, and Canidida.

In another specific embodiment, the non-human organism is a Caenorhabduselegans nematode.

In another specific embodiment, the non-human organism is a plantselected from the group consisting of an apple, Arabidopsis, bajra,banana, barley, beans, beet, blackgram, chickpea, chili, cucumber,eggplant, favabean, maize, melon, millet, mungbean, oat, okra, Panicum,papaya, peanut, pea, pepper, pigeonpea, pineapple, Phaseolus, potato,pumpkin, rice, sorghum, soybean, squash, sugarcane, sugarbeet,sunflower, sweet potato, tea, tomato, tobacco, watermelon, and wheat.

In another specific embodiment, the non-human organism is a Mus musculusmouse.

Measuring Gene Expression/Transcription

One useful measurement of the methods of modulating gene expressionusing the novel polynucleotides, polypeptides, vectors, and/or geneexpression cassettes of the present invention is that of thetranscriptional state of the cell including the identities andabundances of RNA, preferably mRNA species. Such measurements areconveniently conducted by measuring cDNA abundances by any of severalexisting gene expression technologies.

Nucleic acid array technology is a useful technique for determiningdifferential mRNA expression. Such technology includes, for example,oligonucleotide chips and DNA microarrays. These techniques rely on DNAfragments or oligonucleotides which correspond to different genes orcDNAs which are immobilized on a solid support and hybridized to probesprepared from total mRNA pools extracted from cells, tissues, or wholeorganisms and converted to cDNA. Oligonucleotide chips are arrays ofoligonucleotides synthesized on a substrate using photolithographictechniques. Chips have been produced which can analyze for up to 1700genes. DNA microarrays are arrays of DNA samples, typically PCRproducts, that are robotically printed onto a microscope slide. Eachgene is analyzed by a full or partial-length target DNA sequence.Microarrays with up to 10,000 genes are now routinely preparedcommercially. The primary difference between these two techniques isthat oligonucleotide chips typically utilize 25-mer oligonucleotideswhich allow fractionation of short DNA molecules whereas the larger DNAtargets of microarrays, approximately 1000 base pairs, may provide moresensitivity in fractionating complex DNA mixtures.

Another useful measurement of Applicants' methods of the invention isthat of determining the translation state of the cell by measuring theabundances of the constituent protein species present in the cell usingprocesses well known in the art.

Where identification of genes associated with various physiologicalfunctions is desired, an assay may be employed in which changes in suchfunctions as cell growth, apoptosis, senescence, differentiation,adhesion, binding to a specific molecules, binding to another cell,cellular organization, organogenesis, intracellular transport, transportfacilitation, energy conversion, metabolism, myogenesis, neurogenesis,and/or hematopoiesis is measured.

In addition, selectable marker or reporter gene expression may be usedto measure gene expression modulation using Applicants′ invention.

Other methods to detect the products of gene expression are well knownin the art and include Southern blots (DNA detection), dot or slot blots(DNA, RNA), northern blots (RNA), RT-PCR (RNA), western blots(polypeptide detection), and ELISA (polypeptide) analyses. Although lesspreferred, labeled proteins can be used to detect a particular nucleicacid sequence to which it hybridizes.

In some cases it is necessary to amplify the amount of a nucleic acidsequence. This may be carried out using one or more of a number ofsuitable methods including, for example, polymerase chain reaction(“PCR”), ligase chain reaction (“LCR”), strand displacementamplification (“SDA”), transcription-based amplification, and the like.PCR is carried out in accordance with known techniques in which, forexample, a nucleic acid sample is treated in the presence of a heatstable DNA polymerase, under hybridizing conditions, with one pair ofoligonucleotide primers, with one primer hybridizing to one strand(template) of the specific sequence to be detected. The primers aresufficiently complementary to each template strand of the specificsequence to hybridize therewith. An extension product of each primer issynthesized and is complementary to the nucleic acid template strand towhich it hybridized. The extension product synthesized from each primercan also serve as a template for further synthesis of extension productsusing the same primers. Following a sufficient number of rounds ofsynthesis of extension products, the sample may be analyzed as describedabove to assess whether the sequence or sequences to be detected arepresent.

The present invention may be better understood by reference to thefollowing non-limiting Examples, which are provided as exemplary of theinvention.

EXAMPLES

General Molecular Biology Techniques

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

Conventional cloning vehicles include pBR322 and pUC type plasmids andphages of the M13 series. These may be obtained commercially (BethesdaResearch Laboratories).

For ligation, DNA fragments may be separated according to their size byagarose or acrylamide gel electrophoresis, extracted with phenol or witha phenol/chloroform mixture, precipitated with ethanol and thenincubated in the presence of phage T4 DNA ligase (Biolabs) according tothe supplier's recommendations.

The filling in of 5′ protruding ends may be performed with the Klenowfragment of E. coli DNA polymerase I (Biolabs) according to thesupplier's specifications. The destruction of 3′ protruding ends isperformed in the presence of phage T4 DNA polymerase (Biolabs) usedaccording to the manufacturer's recommendations. The destruction of 5′protruding ends is performed by a controlled treatment with S1 nuclease.

Mutagenesis directed in vitro by synthetic oligodeoxynucleotides may beperformed according to the method developed by Taylor et al. [NucleicAcids Res. 13 (1985) 8749-8764] using the kit distributed by Amersham.

The enzymatic amplification of DNA fragments by PCR[Polymerase-catalyzed Chain Reaction, Saiki R. K. et al., Science 230(1985) 1350-1354; Mullis K. B. and Faloona F. A., Meth. Enzym. 155(1987) 335-350] technique may be performed using a “DNA thermal cycler”(Perkin Elmer Cetus) according to the manufacturer's specifications.

Verification of nucleotide sequences may be performed by the methoddeveloped by Sanger et al. [Proc. Natl. Acad. Sci. USA 74: 5463-5467(1977)] using the kit distributed by Amersham.

Plasmid DNAs may be purified by the Qiagen Plasmid Purification Systemaccording to the manufacture's instruction.

Example 1

This Example describes the cloning of full-length cDNA encoding greenleafhopper Nephotetix cincticeps ecdysone receptor polypeptide. Toisolate the full length coding sequence of this previously unknownleafhopper ecdysone receptor isoform (herein named “NcEcR”), Applicantsused degenerate oligonucleotides (see Table 2) designed based onconserved regions at the end of A/B and E domains to amplify and cloneNcEcR from cDNA prepared from RNA obtained from green leafhopper cells.Primer and SEQ ID NO: Primer Nucleic Acid Sequence Primer 1 5′-CAA/GGAA/G GAA/G CTG/C TGT/C (SEQ ID NO: 3) CTG/C GT-3′ Primer 2 5′-TCC CAA/GATC/T TCT/C TCG/C AGG/A (SEQ ID NO: 4) AA-3′

Polymerase chain reaction (PCR) amplification was performed using thePrecision Taq polymerase kit (Stratagene) using the reaction conditionsand cycling parameters as follows. PCR was performed using 1× reactionbuffer (supplied by manufacturer), 50 ng of dsDNA template, 125 ng offorward primer (Primer 1), 125 ng of reverse complementary primer(Primer 2), and 1 μl of dNTP mix (supplied by manufacturer) in a finalreaction volume of 50 μL. The cycling parameters used consisted of onecycle of denaturing at 95° C. for 30 seconds, followed by 35 cycles ofdenaturing at 94° C. for 1 minute, annealing at 50° C. for 1 minute, andextending at 72° C. for 2 minutes, followed by a final elongation cycleat 72° C. for 2 minutes.

The resulting PCR products were cloned into PCRII vector (Invitrogen),sequenced and the full-length coding sequence determined. Thepolynucleotide sequence encoding the full length NcEcR is presented asSEQ ID NO: 1. The deduced amino acid sequence of the full-length NcEcRis presented herein as SEQ ID NO: 2 and showed high similarity with thededuced amino acid sequence of other EcRs. As shown in Table 3, NcEcRshowed highest similarity with locust, whitefly and mealworm EcRs. It isalso interesting that NcEcR showed higher similarity with Crab and TickEcRs when compared to lepidopteran and dipteran insect EcRs.

Applicants have aligned the polypeptide sequences of the NcEcR and EcRsfrom other species and identified the percent amino acid similarity ascompared to the full-length NcEcR The results are presented in Table 3.TABLE 3 Comparison of leafhopper EcR (NcEcR) with other EcRs. PercentAmino Acid EcR Similarity with NcEcR LmEcR 78.3 BaEcR 75.1 TmEcR 75.9AmaEcR 63.1 CpEcR 65.6 AaEcR 62.1 DmEcRB 61.0 LcEcR 62.3 CcEcR 61.2CfEcRB 58.0 BmEcR 55.3 MsEcR 59.1 CtEcR 58.5 FXR 35.0 LXR 37.4 TR 28.7VDR 25.7

Example 2

This Example describes the construction of leafhopper ecdysone receptorgene expression cassettes and their use in an ecdysone receptor-basedgene expression modulation system The results presented hereindemonstrate that a leafhopper ecdysone receptor is functional in anecdysone receptor-based gene expression modulation system in both insectand mammalian cells.

A) Insect Cells:

Briefly, the NcEcR CDE domains (amino acids 6-370 of SEQ ID NO: 2) werefused to a VP16 transactivation domain (SEQ ID NO: 5) as follows. Aconstruct was prepared by fusing a polynucleotide (nucleotides 16-1109of SEQ ID NO: 1) encoding an NcEcR-CDE polypeptide to a polynucleotide(SEQ ID NO: 6) encoding a VP16 activation domain at the NH2 terminalend. This VP16NcEcR fusion was then cloned under the control ofbaculovirus IE1 promoter (SEQ ID NO: 7). The VP16NcEcR gene expressioncassette was transfected into L57 cell line (Drosophila cell line thatlacks endogenous EcR) and a cotton boll weevil (“CBW”; Anthonomusgrandis) BRL-AG-2 cell line (generously provided by USDA, ARS,Bioscience Research Laboratory, Fargo, N.D.) along with a reporterconstruct EcRELaCZ that comprises a 6×ECRE response element (1×EcRE isshown in SEQ ID NO: 8), an ADH distal promoter (see Heberlein et al.,1985, Cell 41: 965-977 and Koelle et al. 1991, Cell 67: 59-77) and aLacZ reporter gene (SEQ ID NO: 9). The reporter gene activity wasquantified in the presence of a series of concentrations of20-hydroxyecdysone (20E) andN′-tert-butyl-N′-(3,5-dimethylbenzoyl)-3-methoxy-2-ethylbenzohydrazide(RH-123709) ligands.

Ligands: The steroid ligand 20-hydroxyecdysone (20E) was purchased fromSigma Chemical Company. The non-steroidal ligandN′-tert-butyl-N′-3,5-dimethylbenzoyl)-3-methoxy-2-ethylbenzohydrazide(RH-123709) is a synthetic stable ecdysteroid ligand synthesized at Rohmand Haas Company. The ligands were dissolved in DMSO and the finalconcentration of DMSO was maintained at 0.1% in both controls andtreatments.

Transfections: DNAs corresponding to the gene constructs described abovewere transfected into L57 or the CBW cells as follows. L57 cells weregrown in HyQ-CCM3 medium (Hyclone labs) and transfected withlipofectomine (Life Technologies). The CBW cells were grown in Ex-Cell401 (JRH Sciences) and transfected with Celfectin (Invitrogen). Standardmethods for culture and maintenance of the cells were followed. Cellswere harvested when they reached 50% confluency and plated in 6-, 12- or24-well plates at 125,000, 50,000, or 25,000 cells, respectively, in2.5, 1.0, or 0.5 ml of growth medium, respectively. The next day, thecells were rinsed with growth medium and transfected for four hours. For12-well plates, 4 μl of the appropriate transfection reagent was mixedwith 100 μl of growth medium One μg of reporter construct and 0.25 μg ofeach receptor construct of the receptor pair to be analyzed were addedto the transfection mix. A second reporter construct was added [pTKRL(Promega), 0.1 μg/transfection mix] that comprises a Renilla luciferasegene operably linked and placed under the control of a thymidine kinase(TK) constitutive promoter and was used for normalization. The contentsof the transfection mix were mixed in a vortex mixer and let stand atroom temperature for 30 minutes. At the end of incubation, thetransfection mix was added to the cells maintained in 400 μl growthmedium. The cells were maintained at 37° C. and 5% CO₂ for four hours.At the end of incubation, 500 μl of growth medium and eitherdimethylsulfoxide (DMSO; control) or a DMSO solution of 0, 0.001, 0.01,0.1, 1.0, 10, or 100 μM steroidal ligand or non-steroidal ligand wasadded and the cells were maintained at 37° C. and 5% CO₂ for 48 hours.The cells were harvested and reporter activity was assayed. The sameprocedure was followed for 6 and 24 well plates as well except all thereagents were doubled for 6 well plates and reduced to half for 24-wellplates.

Reporter Assays: Cells were harvested 48 hours after adding ligand. 125μl of passive lysis buffer (art of Dual-luciferase™ reporter assaysystem from Promega Corporation) were added to each well of the 24-wellplate. The plates were placed on a rotary shaker for 15 minutes. Twentyμl of lysate were assayed. Luciferase activity was measured usingDual-luciferase™ reporter assay system from Promega Corporationfollowing the manufacturer's instructions. β-Galactosidase was measuredusing Galacto-Star™ assay kit from TROPIX following the manufacturer'sinstructions. All luciferase and β-galactosidase activities werenormalized using Renilla luciferase as a standard. Fold activities werecalculated by dividing normalized relative light units (“RLU′) in ligandtreated cells with normalized RLU in DMSO treated cells (untreatedcontrol).

As shown in FIG. 1, the NcEcR construct was able to transactivatereporter gene activity in a dose-dependent manner with both ligandstested in the CBW cells. However, in L57cells there was very littletransactivation in the presence of either ligand. Applicants' previousstudies have shown that both CfEcR and DmEcR cause good transactivationin L57 but NcEcR was a poor transactivator in these cells.

B) Mammalian Cells:

Briefly, the NcEcR DE domains (amino acids 72-370 of SEQ ID NO: 2) werefused to a GAL4 DNA binding domain (SEQ ID NO: 10) as follows. Aconstruct was prepared by fusing a polynucleotide (nucleotides 214-1109of SEQ ID NO: 1) encoding an NcEcR-DE polypeptide to a polynucleotide(SEQ ID NO: 11) encoding a GAL4 DNA-binding domain at the NH2 terminalend. This GAL4/NcEcR fusion was then cloned under the control of acytomegalovirus (CMV) promoter (SEQ ID NO: 12). In addition, apolynucleotide encoding the EF domains of seven RXR/USPs from a mothChoristoneura fumiferana ultraspiracle protein (“CfUSP”, SEQ ID NO: 13),a fruit fly Drosophila melanogaster ultraspiracle protein (“DmUSP”; SEQID NO: 14), a locust Locusta migratoria ultraspiracle protein (LmUSP;SEQ ID NO: 15), a mouse Mus musculus retinoid X receptor isoform α(MmRXRα; SEQ ID NO: 16), a chimeric RXR/USP between MmRXRα and LmUSP(Chimera; SEQ ID NO: 17), a tick Amblyomma americanum retinoid Xreceptor homolog 1 (AmaRXR1; SEQ ID NO: 18), and a tick Amblyommaamericanum retinoid X receptor homolog 2 (AmaRXR2; SEQ ID NO: 19) wereeach fused to a polynucleotide (SEQ ID NO: 6) encoding a VP16 activationdomain.

The GAL4/NcEcR gene expression cassette was transfected into NIH3T3cells (ATCC) along with each of the seven VP16RXR/USP gene expressioncassettes and a reporter construct pFRLuc that comprises a 5XGALARE (1XGALARE is shown in SEQ ID NO: 20), a synthetic TATAA (SEQ ID NO: 21) anda luciferase reporter gene (SEQ ID NO: 22) as described above except thecells were cultured in growth media comprising 10% fetal bovine serum(FBS), Superfect™ (Qiagen Inc.) was used as the transfection reagent,and at the end of incubation/transfection, 500 μl of growth mediumcontaining 20% FBS was added to the cells.

The receptor combinations were compared for their ability totransactivate pFRLuc in NIH3T3 cells in the presence of 0, 0.2, 1.0, or10 μM steroid ligand ponasteroneA (PonA; Invitrogen) or 0, 0.04, 0.2,1.0, or 10 μM GS™-E. The non-steroidal ligandN-(2-ethyl-3-methoxybenzoyl)-N′-3,5-dimethylbenzoyl)-N′-tert-butylhydrazine(GS™-E) is a synthetic stable ecdysteroid ligand synthesized at Rohm andHaas Company. The ligand was dissolved in DMSO and the finalconcentration of DMSO was maintained at 0.1% in both controls andtreatments. The results are presented in FIG. 2 and the numbers on thetop of each bar show the maximum fold induction for that group.

As shown in FIG. 2, NcEcR in combination with MmRXRα, AmaRXR1, orAmaRXR2 supports induction of reporter activity. NcEcR in combinationwith CfUSP, DmUSP, Chimera or LmUSP produced lower fold induction mainlydue to increased background activity of the reporter in the absence ofligand.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1-19. (canceled)
 20. A gene switch that controls expression of a gene ofinterest comprising: a first gene expression cassette comprising apromoter operably linked to a polynucleotide sequence that encodes apolypeptide comprising: a DNA-binding domain that recognizes a responseelement associated with a gene of interest; a ligand binding domaincomprising at least a portion of a ligand binding domain from ahomopteran ecdysone receptor polypeptide.
 21. The gene switch accordingto claim 20, further comprising a second gene expression cassettecomprising: a response element to which the DNA-binding domain of thepolypeptide binds; and a gene of interest.
 22. The gene switch of claim20, wherein the promoter is a tissue-specific promoter.
 23. The geneswitch according to claim 22, wherein the tissue-specific promoter is adevelopmentally-specific promoter.
 24. A vector comprising the geneswitch of claim
 20. 25. A gene switch that controls expression of a geneof interest comprising: a first gene expression cassette comprising afirst promoter operably linked to a polynucleotide sequence that encodesa first polypeptide comprising: i) a DNA-binding domain; and ii) aligand binding domain comprising at least a portion of a ligand bindingdomain from a homopteran ecdysone receptor; and a second gene expressioncassette comprising a second promoter operably linked to apolynucleotide sequence that encodes a second polypeptide comprising:iii) a transactivation domain; and iv) a ligand binding domaincomprising at least a portion of a ligand binding domain from an RXRreceptor or RXR receptor homolog.
 26. The gene switch according to claim25, further comprising a third gene expression cassette comprising: aresponse element to which the DNA-binding domain of the firstpolypeptide binds; a promoter that is activated by the transactivationdomain of the second polypeptide; and a gene of interest.
 27. The geneswitch of claim 25, wherein the first and second promoters are the same.28. The gene switch of claim 25, wherein at least one of the first andsecond promoters is a tissue-specific promoter.
 29. An isolated hostcell into which the gene switch according to claim 20 has beenintroduced.
 30. The isolated host cell according to claim 29, whereinthe host cell is selected from the group consisting of a bacterial cell,a fungal cell, a yeast cell, a plant cell, an animal cell, a mammaliancell, a mouse cell, and a human cell.
 31. A non-human organismcomprising a host cell into which the tissue-specific gene switchaccording to claim 20 has been introduced.
 32. The non-human organismaccording to claim 31, wherein the non-human organism is selected fromthe group consisting of a bacterium, a fungus, a yeast, a plant, ananimal, and a mammal.
 33. A system that controls expression of a gene ofinterest in a host cell comprising, a gene switch, wherein the geneswitch comprises at least a portion of a homopteran ecdysone receptor;and a ligand.
 34. The system of claim 33, wherein the system is used ingene therapy applications.
 35. The system of claim 33, whereinexpression of the gene switch is driven by a tissue-specific promoter.